Monkeypox Virus

I. Organism Information

A. Taxonomy Information
  1. Species:
    1. Monkeypox virus :
      1. GenBank Taxonomy No.: 10244
      2. Description: Human monkeypox is a zoonotic disease caused by the monkeypox virus (MPXV), a member of the genus Orthopoxvirus (family Poxviridae, subfamily Chordopoxvirinae). Other notable members of this group include variola virus (the causative agent of smallpox) and vaccinia virus (the virus used in the smallpox vaccine). Less well-known members include ectromelia, camelpox, and cowpox viruses (Nalca et al., 2005). It is a sporadic smallpox-like zoonotic viral exanthema that occurs in the rain forests of Central and West Africa (Hutin et al., 2001). Human monkeypox is clinically almost identical to ordinary smallpox, and therefore, since the global eradication of smallpox in 1977, much attention has been paid to monkeypox as a smallpox-like disease and possible agent of bioterrorism. Additional attention was brought to bear on this virus when, in the spring of 2003, it emerged for the first time in the Western Hemisphere and caused a cluster of cases in the US Midwest (Nalca et al., 2005).
      3. Variant(s):
        • Callithrix jacchus orthopoxvirus :
          • GenBank Taxonomy No.: 243803
          • Parent: Monkeypox virus
          • Description: An epizootic poxvirus infection that occurred in a colony of 80 common marmosets (Callithrix jacchus) introduced to a laboratory facility (Gough et al., 1982).
        • Monkeypox virus (strain Sierra Leone 70-0266) :
B. Lifecycle Information :
  1. Virion :
    1. Size: Poxviridae virus family, i.e., unusually large brick-shaped viruses of approximately 270 x 350 nm size (Berting et al., 2005).
    2. Shape: Characteristic poxvirus virions showing the typical morphology (i.e., brick shape with lateral bodies and a central core) would be expected to be observed under electron microscopy (Nalca et al., 2005).
C. Genome Summary:
  1. Genome of Monkeypox virus
    1. Description: Monkeypox virus, complete genome (NCBI Entrez)
    2. Chromosome:
      1. GenBank Accession Number: NC_003310
      2. Size: 196,858 nt (NCBI Entrez)
      3. Gene Count: 191 genes (NCBI Entrez)
      4. Description: The 196 858 bp of MPV-ZAI DNA, comprising the entire genome contains 190 largely non-overlapping ORFs of 60 or >60 amino acid residues as well as structural features and a GC content of 31.1% similar to other orthopoxviruses. The nucleotide sequence within the central region of the MPV genome (MPV isolate from a patient during a large human monkeypox outbreak in Zaire in 1996), which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome (Shchelkunov et al., 2001).

II. Epidemiology Information

Monkeypox virus was first identified as the causal agent of a pox infection in captive cynomolgus monkeys (Macaca fascicularis) in Copenhagen, Denmark, in 1958. Eight more outbreaks occurred during the next 10 years in the United States and the Netherlands among groups of captive monkeys imported from Malaysia, India, and the Philippines. Although monkeypox virus was recovered from captive primates originally collected from these Asian areas, there is no virologic, serologic, or epidemiologic evidence that the virus occurs naturally anywhere outside of Africa. Although the definitive natural reservoir is still unknown, studies point to rope squirrels of the African genus, Funisciurus. Primates, rabbits, and several rodent species are also vulnerable to infection (Sale et al., 2006). Monkeypox (MPX) was first identified as a human pathogen in 1970 in Zaire, now the Democratic Republic of the Congo. Since then, sporadic human MPX outbreaks have occurred in forested regions of central and west Africa, with most cases occurring in the Congo River Basin. The clinical presentation of this disease in humans resembles smallpox, and human cases of MPX in central Africa have been reported to be more severe than those in west Africa. Morbidity and mortality of the disease have been highly variable in African outbreaks, with case fatality rates ranging from 1% to 20% and a significant secondary transmission rate (Sbrana et al., 2007). Some cases can be exceedingly mild or atypical and may easily remain undetected and unreported. Pronounced lymphadenopathy has been the only clinical feature found commonly in monkeypox but not in smallpox. Smallpox vaccination protects against monkeypox (Jezek et al., 1983). Human monkeypox, occurring in the tropical rainforest of west and central Africa, is regarded as the most important orthopoxvirus infection for epidemiological surveillance during the post-smallpox era. Fifty-seven cases of human monkeypox have occurred since 1970, in the tropical rainforests in six west and central African countries, the majority of them (45) being reported from Zaire. The disease appears to be more frequent in dry season. Children below ten years of age comprise 84% of the cases. Clusters of cases have been observed in certain areas within countries and within affected households. Human-to-human spread has possibly occurred seven times. No cases of possible tertiary spread were observed (Jezek et al., 1983). Human monkeypox is the most important orthopoxvirus infection in the post-smallpox eradication period. The disease is a zoonosis and person-to-person transmission is rather difficult. Thus, this episode is a rare event and special analysis of the circumstances is discussed. However, it supports the necessity to carry out surveillance and research on this disease (Jezek et al., 1986). Surveillance in Zaire (now the Democratic Republic of the Congo (DR Congo)) from 1981 to 1986 indicated that most of the cases were primary animal-to-human (72% of cases) and that the disease occurred mainly in young children not vaccinated against smallpox (smallpox vaccination effectively protects against MPV infection). The study showed the household secondary human-to-human attack rate of MPV was relatively low, 4-12% in unvaccinated persons compared with smallpox, which had a rate of approximately 70%. The animal reservoir for human infections is still poorly understood; however, arboreal squirrels are prime suspects, and monkeys, like humans, appear to be sporadically infected. Recently an outbreak of human monkeypox was recognized in the Katako-Kombe and Lodja Health Zones of the Kasai Oriental Province of DR Congo. Surveillance showed 501 suspected cases in approximately 300 000 persons over 2 years time with peaks of disease in August 1996 and 1997. Seventy-eight percent of cases were from human-to-human transmission; however, the household secondary attack rate was similar to the rate in unvaccinated persons in the 1981-1986 surveillance. Several isolates of MPV were recovered during the 1996-1997 surveillance. The approximately 8% secondary attack rate and limited sequence analysis have indicated little change in MPV compared with isolates from the 1980s, though case fatalities in the 1980s approached 10% and current case fatalities are 1.5%. Laboratory studies of serum samples available for approximately 360 of the 501 suspected cases indicated 60% (approximately 215) had orthopoxvirus antibodies and the remaining 40% may have been cases of varicella chickenpox (Neubauer et al., 1998). The WHO's Global Commission for the Certification of Smallpox Eradication declared human monkeypox the most important orthopoxvirus infection of human beings in the post-smallpox era. As monkeypox surveillance programmes intensified during the 1980s, the incidence of this seemingly rare disease increased. Although the documented rates of transmissibility and mortality of human monkeypox appear to have changed dramatically from the 1970s to the 1990s, careful analysis suggests that these changes are an artefact caused by the use of variable case definitions in different epidemiological settings (passive vs active surveillance, outbreak investigations). Data from the 1981-86 active surveillance programme seem to be the most precise. Once limited to the remote rainforests of central and western Africa, human monkeypox has now emerged in North America with the recent introduction of the virus through infected exotic pets. This event was predictable given US importation practices. Three of the six genera of rodents represented in the contaminated African shipment were previously documented to have high seroprevalence rates for MPV in environmental surveys. Whether MPV has established an enzootic reservoir in the USA remains to be seen. If it has, the public-health consequences will be difficult to predict from the accumulated African data owing to potential differences in population density, immunity, and rates of immunosuppression, particularly if the US reservoir includes wild rodents in an urban setting (Di Giulio and Eckburg, 2004).

A. Outbreak Locations:
  1. USA: During May and June 2003, the first cluster of human monkeypox cases in the United States was reported. Most human case-patients with this febrile vesicular rash illness were believed to have acquired the infection from prairie dogs (Cynomys spp.) that became ill after contact with various exotic African rodents (Funiscuirus spp., Heliosciurus spp., Cricetomys spp., Atherurus spp., Graphiurus spp., and Hybomys spp.) shipped from Ghana to the United States in April 2003 (Guarner et al., 2004).
  2. Zaire: An outbreak of five cases of human monkeypox occurred in children belonging to two families living in the West Kasai region of Zaire during May-July 1983. Epidemiologic investigations suggest that the first case was infected from an animal source, possibly a monkey, and that each of the other four cases was infected from a previous human case. Three of these cases of presumed person-to-person transmission occurred in close household contacts. The other case infection occurred either by casual contact within the hospital compound, or possibly because of infection due to use of the same syringe for injections (Jezek et al., 1986). An outbreak was reported in Katako-Kombe in the Sankaru Sub-region of Kasai Oriental Region during July and August 1996. The first cases occurred in mid-February 1996, but the outbreak was not apparent and reported until the end of July when more people became infected. Seventy-one cases, of which 6 were fatal, was reported in 13 villages (total population of 15698) (World Health Organization, 1996). Seven outbreaks of disease characterized by a pustular rash and suspected to have been caused by human monkeypox virus were investigated. The outbreaks occurred between February and August 2001 in the province of Equateur in the Democratic Republic of Congo. The outbreaks involved a total of 31 persons and caused five deaths. Specimens from 14 patients were available and were analyzed by electron microscopy, virus isolation, and PCR assays specific for monkeypox virus and varicella-zoster virus. Two outbreaks were indeed caused by monkeypox virus (16 cases, with four deaths), and that in two outbreaks both monkeypox and varicella-zoster virus were involved (seven cases, with one death) (Meyer et al., 2002).
  3. West Africa: Between October 1970 and May 1971, six cases of human infection with monkeypox virus were identified in three West African countries, Liberia, Nigeria, and Sierra Leone. Four of the cases were confirmed by virus isolation and two were diagnosed on the basis of epidemiological and serological investigations (Foster et al., 1972).
B. Transmission Information:
  1. From: Human To: Human
    Mechanism: Human-to-human transmission occurs with an incubation period of 12 days (range 7-21 days) (Hutin et al., 2001). Like variola virus, MPXV (Congo basin variant) can be spread from person to person, although it is evident that interhuman spread of MPXV is less efficient than was historically observed for variola virus. Person-to-person spread of both viruses is thought to occur principally via infectious oropharyngeal exudates generated during the rash phase of illness or possibly during the preceding 2-3-day period of febrile prodrome (Reynolds et al., 2006). It is also transmitted from human-to-human by respiratory droplets or body fluids (Sale et al., 2006). Secondary transmission was determined to be the most likely cause of infection in 4 cases in Zaire (the present Democratic Republic of Congo [DRC]), with secondary attack rates of 7.5% among close family members living in the same household and 3.3% among all susceptible contacts (Nalca et al., 2005).

  2. From: Animal To: Human
    Mechanism: Like vaccinia virus and cowpox virus, MPXV can infect a broad range of mammalian animal species and can be transmitted to humans by means of direct contact with infected animals, often by means of traumatic injury to the skin (Reynolds et al., 2006). All cases in the DRC occurred in areas bordering tropical rain forests and appeared to be associated with animal contact (Nalca et al., 2005). A consistently high level of antibody prevalence found among squirrels Heliosciurus rufobrachium suggested that this species is also steadily involved in the transmission. The squirrels, the only mammals frequently infected by monkeypox in the areas of human activities, which are frequently trapped by the population, are obviously a major source of infection for human beings (Khodakevich et al., 1987).

C. Environmental Reservoir:
  1. Squirrel :
    1. Description: Animal antibody surveys in the Democratic Republic of Congo (DRC; former Zaire) suggested that squirrels play a major role as a reservoir of the virus and that humans are sporadically infected (Hutin et al., 2001). The primary reservoir for human infection, however, remains unknown. Several epidemiological studies from the Democratic Republic of Congo have implicated squirrels (especially Funisciurus anerythrus) inhabiting agricultural areas as primary candidates to sustain viral transmission among people in nearby settlements. In one environmental survey, Funisciurus spp squirrels had a higher rate of MPV seropositivity (24%) than other animals that were tested, including Heliosciurus spp squirrels (15%) and primates (8%). A subsequent seroprevalence study done as part of the investigation of the outbreak in February, 1997, in the Democratic Republic of Congo showed even higher positivity rates in these squirrels (39-50% in Funisciurus spp and 50% in Heliosciurus spp squirrels) (Di Giulio and Eckburg, 2004). A high prevalence of monkeypox-specific antibodies (24.7%) found in 320 squirrels of Funisciurus anerythrus species in the Bumba zone of Zaire during a survey in January-February 1986, suggested that these animals sustain virus transmission in the areas surrounding human settlements. A consistently high level of antibody prevalence found among squirrels Heliosciurus rufobrachium suggested that this species is also steadily involved in the transmission. The squirrels, the only mammals frequently infected by monkeypox in the areas of human activities, which are frequently trapped by the population, are obviously a major source of infection for human beings (Khodakevich et al., 1987).
  2. Monkey :
    1. Description: Animal antibody surveys in the Democratic Republic of Congo (DRC) suggested that squirrels and monkeys play a role in the life cycle of the virus (Meyer et al., 2002).
  3. Rodent :
    1. Description: The reservoir for MPXV is still unknown. However, there are data to suggest that the reservoir is likely to be 1 or numerous species of rodents or squirrels that inhabit the secondary forest of central Africa (Nalca et al., 2005). In addition, 16% of Gambian giant rats tested in a study had serological evidence of MPV exposure (Di Giulio and Eckburg, 2004).
  4. Prairie dogs :
    1. Description: Virologic evidence from North American prairie dogs (Cynomys sp.) was concordant with their suspected roles as vectors for human monkeypox. Multiple tissues were found suitable for DNA detection and/or virus isolation. These data extend the potential host range for monkeypox virus infection and supports concern regarding the potential for establishment in novel reservoir species and ecosystems (Hutson et al., 2007).
D. Intentional Releases:
  1. Intentional Release information :
    1. Description: There are no data available on intentional release of Monkeypox virus. Although, MPV has been described as having a low potential for use as an agent of biological warfare, this may no longer be the case in the era of modern molecular biology (Di Giulio and Eckburg, 2004).
    2. Emergency contact: All suspected cases of human monkeypox should be immediately reported to a local health department. Although clinical characteristics can be helpful in differentiating various poxvirus infections from other causes of vesiculopustular rashes laboratory confirmation is required for definitive diagnosis (Di Giulio and Eckburg, 2004).
    3. Containment: Human monkeypox has been a relatively newly recognized disease. Studies are in progress to identify the natural cycle of monkeypox virus and to define better its clinical and epidemiological characteristics. Special surveillance is maintained in endemic areas with the aim to provide assurance that in spite of waning immunity of the human population following cessation of the smallpox vaccination, the disease does not constitute a potential danger to man (Jezek et al., 1983). It is predicted that monkeypox virus will continue to be introduced into human communities from animal sources, and that the average magnitude and duration of monkeypox epidemics will increase as vaccine-derived protection declines in the population. On the other hand, current evidence indicates that the virus is appreciably less transmissible than was smallpox, and that it will not persist in human communities, even in the total absence of vaccination. The findings thus support the recommendation of the Global Commission for the Certification of Smallpox Eradication to cease routine smallpox vaccination in monkeypox endemic areas, but to encourage continued epidemiological surveillance (Fine et al., 1988).

III. Infected Hosts

  1. Man:
    1. Taxonomy Information:
      1. Species:
        1. Human, man :
          • GenBank Taxonomy No.: 9606
          • Scientific Name: Homo sapiens (NCBI Taxonomy)
          • Description: Human monkeypox virus appears to be the result of chance infection in man with an animal virus. The natural reservoir, pattern of transmission, and route of infectivity remain unknown (Foster et al., 1972). Human monkeypox is clinically almost identical to ordinary smallpox, and therefore, since the global eradication of smallpox in 1977, much attention has been paid to monkeypox as a smallpox-like disease and possible agent of bioterrorism. Additional attention was brought to bear on this virus when, in the spring of 2003, it emerged for the first time in the Western Hemisphere and caused a cluster of cases in the US Midwest (Nalca et al., 2005).

    2. Infection Process:
      1. Infectious Dose: There are no quantitative data on potential dose and specific exposure types (Sejvar et al., 2004).
      2. Description: During the first week of the rash, the patient is considered to be infectious and should be isolated until all scabs separate and results of throat swab PCR are negative (Nalca et al., 2005).

    3. Disease Information:
      1. Monkeypox (i.e., Human monkeypox) :
        1. Pathogenesis Mechanism: After an incubation period of 7-19 days, clinical monkeypox is characterized by a prodrome of fever, headache, and fatigue. Adenopathy is common and may distinguish this infection from smallpox. Generally, the rash evolves uniformly as macules, papules, vesicles, and pustules and then crusts over the course of 2-3 weeks. Lesions are most prominent on the head and extremities and often involve the palms and soles. It is unknown whether this is a clinical feature associated with this strain of monkeypox virus or whether skin pigmentation may be responsible for this observation. The lesions of monkeypox may be differentiated from those caused by VZV, a virus also associated with encephalitis. VZV lesions, in contrast to those of monkeypox, are small, superficial, concentrated mainly on the trunk, and classically evolve in different stages (crops). Encephalitis has rarely been associated as a complication of monkeypox infection, having been described only once previously. Vaccinia virus, a related orthopoxvirus, has been associated with a complication known as postvaccinial encephalomyelitis (PVE) when administered as vaccination against smallpox. Cases of PVE have variably displayed clinical and diagnostic features suggestive of a postimmunization demyelinating encephalomyelitis (acute disseminated encephalomyelitis) or direct viral infection of the central nervous system (CNS) . In one patient, who had monkeypox-specific IgM antibodies in CSF, the diagnosis of monkeypox-associated encephalitis was confirmed by skin biopsy and encephalopathy. Since IgM does not normally cross the blood-brain barrier, detection of IgM in CSF is suggestive of active infection of the CNS with intrathecal antibody production and, although dependent on the timing of testing relative to disease progression, serves as the laboratory diagnostic standard for several infectious encephalitides. The presence of intrathecal IgM, cytotoxic changes demonstrated by diffuse and focal edema, and the absence of demyelination all support the association of monkeypox as the cause of acute encephalitis. Weakly positive HHV-2 IgM, generally associated with genital herpes infections, was observed in CSF from the patient; however, this result is discordant with the more-sensitive nonreactive HHV-1 and -2 PCR testing on CSF. Additionally, although the patient had been treated with high-dose acyclovir, clinical features pathognomonic for herpes simplex virus encephalitis, including signal abnormality or hemorrhage of the temporal lobe by neuroimaging and periodic lateralizing epileptiform discharges by EEG, were absent, and no HHV-1 or -2 seroreactivity was seen up to 2.5 weeks into her illness (Sejvar et al., 2004).


        2. Incubation Period: Human-to-human transmission occurs with an incubation period of 12 days (range 7-21 days) (Hutin et al., 2001).


        3. Prognosis: In contrast to variola major, the prognosis of human monkeypox depended largely on the presence or absence of severe complications, which in turn depended on several factors, including vaccination status and concurrent illnesses, such as measles, malaria or diarrheal diseases. The only fatal cases of monkeypox that have been reported occurred in Zaire, where there were 33 deaths among 338 patients between 1981 and 1986, i.e. a crude case-fatality rate of 9.8%. All these deaths occurred among unvaccinated children between 3 months and 8 years of age, giving an overall specific case-fatality rate of 11.2%. The highest case-fatality rate (20%) was found among unvaccinated girls in the 0- to 2-year age group, and the age specific case-fatality rate for the 0- to 4-year age group (15%) was twice that in children in the 5- to 9-year age group (6.6%). The case-fatality rate of unvaccinated primary and co-primary cases (11.8%) did not differ significantly from that found among secondary cases (9.6%). Twenty-one percent of those who died succumed within the first week of illness. 52% during the second week and the remaining 27% during the third week after onset of the rash. No clustering of deaths was observed, except for one episode when deaths of siblings of three age 3 months, 6 months and 3 years were reported in an outbreak in which 4 children were affected in the same household by presumed person-to-person transmission (Jezek and Fenner, 1988).


        4. Diagnosis Overview: Because the clinical picture of monkeypox is very similar to that of chickenpox and that of smallpox, definitive diagnosis is key to keeping natural disease under control or in the early detection of a potential bioterrorism event. Although diseases such as orf and bovine stomatitis (which are caused by parapoxviruses) can produce localized skin lesions similar to those seen in the US monkeypox outbreak, they can be easily distinguished from orthopoxviruses by electron microscopy (Nalca et al., 2005). Clinicians should consider monkeypox in the differential diagnosis of atypical vesiculopustular rash illness, including cases of encephalitis associated with rash. The differential diagnosis of encephalitis in patients with rash should include the common causes of rash-associated viral encephalitis, particularly varicella and HHV infection, but, given our report, should be broadened to include monkeypox in the appropriate context, such as the presence of atypical rash illness, exposure to exotic pets, or recent travel to Africa (Sejvar et al., 2004). Although clinical characteristics can be useful in distinguishing poxvirus infections from other causes of vesiculopustular rashes, laboratory confirmation is required for a definitive diagnosis. The various laboratory diagnostic assays for monkeypox include virus isolation and electron microscopy, PCR, IgM and IgG ELISA, immunofluorescent antibody assay, and histopathologic analysis. Unfortunately, many of these methods are relatively nonspecific and are unable to differentiate MPXV infection from infection with other poxviruses. For example, histologically, the lesions of monkeypox are similar to other viral exanthems (such as those due to variola, cowpox, varicella-zoster, and herpes simplex viruses) and include ballooning degeneration of keratinocytes, prominent spongiosis, dermal edema, and acute inflammation. However, immunohistochemistry analysis, including use of either polyclonal or monoclonal antibodies against all orthopoxviruses, can differentiate between a herpes virus and poxvirus infection. Electron microscopy has often played a major role in viral diagnosis in the past. Likewise, if available, electron microscopy can be a first-line method for laboratory diagnosis of poxvirus infections and may provide one of the first clues to the cause of an unknown rash illness. Characteristic poxvirus virions showing the typical morphology (i.e., brick shape with lateral bodies and a central core) would be expected to be observed under electron microscopy. For example, during the recent US outbreak of monkeypox, lesions viewed by means of electron microscopy showed keratinocytes with large numbers of mature virons, as well as immature virons in the process of assembly (also known as "viral factories") within the cytoplasm. This method, however, cannot differentiate orthopoxvirus species (Nalca et al., 2005). A range of novel diagnostic methods-including PCR, serologic testing, and IHC testing-can be used to confirm various orthopoxvirus infections, including monkeypox (Sejvar et al., 2004). Virus isolation (which can be accomplished by growing the virus in mammalian cell culture) and characterization by various PCR techniques, followed by restriction fragment-length polymorphism analysis or sequencing of amplicons, are often considered to be definitive for the identification of MPXV. In addition, the availability of various real-time PCR assays that use panorthopoxvirus or MPXV-specific targets has increased in recent years. A DNA oligonucleotide microarray with the TNF receptor gene crmB has also been developed as another rapid method for species-specific detection of orthopoxviruses. In contrast to regions outside of Africa, where it is essential to distinguish between monkeypox and a deliberate introduction of smallpox, the principal diagnostic problem in sub-Saharan Africa is to differentiate monkeypox from varicella. During active disease, laboratory confirmation can be performed by PCR analysis of vesicle fluid or scabs, whereas after disease resolution, testing of convalescent-phase serum specimens for anti-varicella virus IgM can be performed. The finding of antipoxvirus antibodies in an unvaccinated individual with a history of severe illness and rash suggests a diagnosis of monkeypox (Nalca et al., 2005). Anti-orthopoxvirus IgM reactivity in CSF may be useful in diagnosing encephalitis due to orthopoxviruses. With further validation, the demonstration of seroconversion to IgM and then an anti-orthopoxvirus IgG response will be a useful tool in diagnosing infectious orthopoxvirus illness (Sejvar et al., 2004).


        5. Symptom Information :
          • General Description: Monkeypox has a clinical presentation very similar to that of ordinary forms of smallpox, including flu-like symptoms, fever, malaise, back pain, headache, and characteristic rash. Given this clinical spectrum, differential diagnosis to rule out smallpox is very important (Nalca et al., 2005). Unlike smallpox, human monkeypox behaves like a classic zoonosis in that most cases represent primary infection from an animal source, and the causative agent appears incapable of sustained secondary transmission in human beings. When infection in human beings does occur, it can be clinically indistinguishable from smallpox, chickenpox, and other causes of a vesiculopustular rash. The most helpful distinguishing clinical feature of human monkeypox is severe lymphadenopathy, however, laboratory testing is necessary for definitive diagnosis (Di Giulio and Eckburg, 2004).
          • Syndrome -- Prodromal illness:
            • Description: After a 10-14-day incubation period, prodromal illness with fever, malaise, and swollen lymph nodes is observed in most of the patients before the development of rash. Lymphadenopathy, which has been observed in 90% of unvaccinated patients, is not a common feature of smallpox and is therefore considered to be a key distinguishing feature of monkeypox. Lymph node enlargement can occur in the submandibular and the cervical or inguinal regions. The prodromal period generally lasts 1-3 days before the occurrence of the typical maculopapular rash (Nalca et al., 2005).


            • Symptoms Shown in the Syndrome:

            • Fever (Nalca et al., 2005):
            • Malaise (Nalca et al., 2005):
            • swollen lymph nodes:
            • Rash:
              • Description: The mean diameter of the skin lesions is 0.5-1 cm, and the clinical progress is very similar to that of ordinary smallpox lesions. During a 2-4-week period, lesions progress from macules to papules, vesicles, and pustules, followed by umbilication, scabbing, and desquamation. Although the rash starts mainly on the trunk, it can spread in a peripheral distribution to the palms and soles of the feet. Lesions can be observed on mucous membranes, in the mouth and tongue, and on the genitalia. In addition to skin lesions, extracutaneous manifestations, such as secondary skin and/or soft-tissue infection (19% of cases), pneumonitis (12%), ocular complications (4%-5%), and encephalitis (<1%) can be observed in patients infected with MPXV (Nalca et al., 2005).
          • Back pain:
            • Description: Other signs and symptoms of monkeypox include chills and/or sweats, headache, backache, sore throat, cough, and shortness of breath (Nalca et al., 2005).
          • Chills (Nalca et al., 2005):
          • Sweats (Nalca et al., 2005):
          • Cough (Nalca et al., 2005):
          • Headache (Nalca et al., 2005):
          • Shortness of breath:
            • Description: Monkeypox virus can be transmitted to humans and cause a syndrome clinically similar to smallpox (e.g., pustular rash, fever, respiratory symptoms such as shortness of breathe, and in some cases, death) (Centers for Disease Control and Prevention (CDC), 1997).
          • Sore throat (Nalca et al., 2005):

        6. Treatment Information:
          • Vaccination: Vaccinia immunisation is roughly 85% effective in preventing human monkeypox, but there is no currently proven treatment for the disease (Di Giulio and Eckburg, 2004). Vaccination combined with an aggressive surveillance program ultimately resulted in the global eradication of smallpox. Unfortunately, eradication of monkeypox is not possible because of the existence of an animal reservoir. However, vaccination with vaccinia virus (the smallpox vaccine) is highly protective against infection with MPXV. In terms of postexposure treatment, vaccination within 4 days after initial close contact with a confirmed monkeypox case is recommended by the Centers for Disease Control and Prevention; however, vaccination should be considered up to 14 days after exposure (Nalca et al., 2005).
            • Applicable: There are no licensed therapies for human monkeypox; however, the smallpox vaccine can protect against the disease (Nalca et al., 2005). In 1968, investigators first reported that monkeys could be immunised against monkeypox by smallpox vaccination. In a later analysis of 215 cases of human monkeypox (209 laboratory confirmed), Fine and colleagues calculated that previous smallpox vaccination, as defined by presence of vaccination scar, conferred 85% protection against monkeypox. Currently, the CDC recommends pre-exposure smallpox vaccination for field investigators, veterinarians, animal-control personnel, and health-care workers who are investigating or caring for patients with suspected monkeypox and who have no contraindications to vaccination (Di Giulio and Eckburg, 2004).
            • Contraindicator: Smallpox vaccination is contraindicated in a person with severe immunodeficiency in T cell function (Nalca et al., 2005).
            • Success Rate: Smallpox vaccine affords protection against monkeypox both by preventing disease and by decreasing disease severity. In the African series, severe disease (e.g., that requiring intensive nursing care or that with the presence of 199 rash lesions) was more common among persons not vaccinated with smallpox vaccine (73.9%) than among vaccinated persons (39.5%). In contrast, milder disease (that without physical incapacitation or that with the presence of <25 rash lesions) was more common among persons vaccinated with smallpox vaccine (37.2%) than among unvaccinated persons (7.5%). These studies demonstrated 85% protection with smallpox vaccine against disease acquisition within households. This is consistent with the mild clinical course of a male patient, the only member of a family of three to have received smallpox vaccination as a child. The unvaccinated mother and child, on the other hand, developed "severe" disease, by the African criteria (Sejvar et al., 2004).
          • Antiviral treatment: Cidofovir: Cidofovir is an antiviral medication with documented in vitro activity against poxviruses, including vaccinia and monkeypox it has been recommended as a second-line agent in the management of possible smallpox vaccine adverse events and may be an agent for consideration in the treatment of monkeypox (Sejvar et al., 2004).
            • Applicable: There is currently no US Food and Drug Administration-approved treatment for monkeypox; however, several options may be of potential benefit (Sejvar et al., 2004).
            • Contraindicator: Cidofovir should not be used for prophylaxis because it has substantial toxic effects (Di Giulio and Eckburg, 2004).
            • Success Rate: Cidofovir is a broad-spectrum antiviral agent with in-vitro activity against virtually all DNA viruses, including MPV. Although this drug has been shown to have in-vivo activity against orthopoxviruses in animals and human beings, no published data are available on its effectiveness for the treatment of human monkeypox. CDC guidelines state that the use of cidofovir can be considered in severe cases of human monkeypox infection. (http://www.cdc.gov/ncidod/monkeypox/treatmentguidelines.htm) (Di Giulio and Eckburg, 2004).
          • vaccinia immune globulin (VIG): The administration of vaccinia immune globulin (VIG) may have efficacy in the treatment of monkeypox, on the basis of serologic cross-protection among the orthopoxviruses (Sejvar et al., 2004).
            • Applicable: Under an investigational new drug protocol, VIG is recommended as treatment for severe adverse events of vaccinia used as smallpox vaccine. It may have efficacy in treatment of complications of monkeypox attributable to viral replication (Sejvar et al., 2004).
            • Success Rate: No data are available on the effectiveness of vaccinia immune globulin (VIG) in the treatment of monkeypox complications. Use of VIG can be considered in severe cases of human monkeypox, although whether it provides any benefit in this setting is unknown. VIG can be considered for prophylaxis in an exposed person with severely impaired cellular immunity for whom smallpox vaccination is contraindicated (Di Giulio and Eckburg, 2004).

        7. Other Information:
          • Supportive care: In some patients, recovery followed supportive care alone (Sejvar et al., 2004). In the USA, clinical consultation on the use of VIG and cidofovir is available from staff at each state health department in the affected area. Current CDC recommendations on the use of vaccinia immunisation, cidofovir, and VIG in people exposed to or infected with MPV are available at http://www.cdc.gov/ncidod/monkeypox/smallpoxvaccine_mpox.htm and http://www.cdc.gov/ncidod/monkeypox/treatmentguidelines.htm (Di Giulio and Eckburg, 2004).

    4. Prevention:
      1. Immunization:
        • Description: Vaccination with vaccinia virus (the smallpox vaccine) is highly protective against infection with MPXV (Nalca et al., 2005).
        • Efficacy:
          • Rate: Monkeypox was not recognized as a distinct disease until 1970, when the elimination of smallpox from Zaire (the present Democratic Republic of Congo [DRC]) revealed the continued occurrence of a smallpox-like illness in rural areas. Widespread vaccination in central Africa during the global eradication campaign presumably caused a temporary reduction in the incidence of human monkeypox, but the absence of immunity in the generation born since that time and the increased dependence on hunting for food in areas devastated by civil war have resulted in reemergence of the disease (Nalca et al., 2005).
          • Duration:
      2. Limiting exposure to infected patients:
        • Description: Effective prevention relies on limiting the contact with infected patients or animals and limiting the respiratory exposure to infected patients (Nalca et al., 2005).

    5. Model System:
      1. Rabbits:
        1. Model Host: Rabbit
        2. Model Pathogens:
        3. Description: After intravenous inoculation, adult rabbits developed systemic disease with a generalized postular rash, conjunctivitis and rhinitis, and the skin lesions sometimes became hemorrhagic before scabbing. Rabbits inoculated by scarification developed a localized papulopustular eruption, whereas intradermal inoculation induced a large indurated lesion with a hemorrhagic center. Generalized lesions also occurred in some of these animals. Young rabbits (<10 days old) were much more susceptible than adult rabbits and developed a generalized disease with a rash, which was usually fatal, even after infection per os or by the intranasal route. Infection could be transmitted by contact from young rabbits infected orally or intranasally to uninfected baby rabbits within the same litter, or even in other cages in the same room (Jezek and Fenner, 1988).
      2. Ground squirrel:
        1. Model Host: Ground squirrel
        2. Model Pathogens:
        3. Description: A proposed new small-animal (rodent) model for studying the pathogenesis and treatment of severe orthopoxvirus infections is described. Thirteen-lined ground squirrels (Spermophilus tridecemlineatus) were infected intraperitoneally and intranasally with monkeypox virus (MPXV). A fulminant illness developed in all animals, and they died 6-9 days after infection. Virus was cultured from the blood and oropharynx several days before death; at necropsy, all of the organs tested contained relatively high titers of MPXV. The major pathologic findings were in the liver, which showed centrilobular necrosis, steatosis, and basophilic inclusion bodies in hepatocytes. Splenic necrosis was also observed, as well as interstitial inflammation in the lungs. The pathologic features of MPXV in ground squirrels are similar to that described with MPXV in macaques and severe variola (smallpox) virus infection in humans (Tesh et al., 2004). The clinical and pathologic characteristics of experimental infection with representative central African and North American MPX virus strains were compared in a ground squirrel model of the disease. The results indicate that the US 2003 virus, which phylogenetically is a member of the west African MPX virus clade, was less virulent than central African MPX virus strains (Sbrana et al., 2007).
      3. Cynomolgous monkeys:
        1. Model Host: Cynomolgous monkeys
        2. Model Pathogens:
        3. Description: Vaccinia-specific B-cell responses are essential for protection of macaques from monkeypox virus. Antibody-mediated depletion of B cells, but not CD4+ or CD8+ T cells, abrogated vaccine-induced protection from a lethal intravenous challenge with monkeypox virus. In addition, passive transfer of human vaccinia-neutralizing antibodies protected non-immunized macaques from severe disease (Edghill-Smith et al., 2005). Modified vaccinia virus Ankara (MVA), a highly attenuated replication-deficient strain of vaccinia virus (VV), has been proven to be safe in humans and immunocompromised animals. The efficacies of MVA alone and in combination with classical VV-based vaccines in a cynomolgus macaque monkeypox model were compared. The MVA-based smallpox vaccine protected macaques against a lethal respiratory challenge with monkeypox virus (Stittelaar et al., 2005). The highly attenuated modified vaccinia virus Ankara (MVA) was compared with the licensed Dryvax vaccine in a monkey model. After two doses of MVA or one dose of MVA followed by Dryvax, antibody binding and neutralizing titres and T-cell responses were equivalent or higher than those induced by Dryvax alone. After challenge with monkeypox virus, unimmunized animals developed more than 500 pustular skin lesions and became gravely ill or died, whereas vaccinated animals were healthy and asymptomatic, except for a small number of transient skin lesions in animals immunized only with MVA (Earl et al., 2004). Cynomolgus monkeys (Macaca fascicularis) were exposed by fine-particle aerosol to lethal doses of monkeypox virus, Zaire strain. Death, attributable to fibrinonecrotic bronchopneumonia, occurred 9 to 17 days postexposure. Lower airway epithelium served as the principal target for primary infection. The relative degree of involvement among lymphoid tissues suggested that tonsil, mediastinal, and mandibular lymph nodes were also infected early in the course of the disease, and may have served as additional, although subordinate, sites of primary replication. The distribution of lesions was consistent with lymphatogenous spread to the mediastinal lymph nodes and systemic dissemination of the virus through a monocytic cell-associated viremia. This resulted in lesions affecting other lymph nodes, the thymus, spleen, skin, oral mucosa, gastrointestinal tract, and reproductive system. The mononuclear phagocyte/dendritic cell system was the principal target within lymphoid tissues and may also have provided the means of entry into other systemic sites. Hepatic involvement was uncommon. Lesions at all affected sites were characterized morphologically as necrotizing. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining of select lesions suggested that cell death within lymphoid and epithelial tissues was due in large part to apoptosis. Skin and mucosal surfaces of the respiratory and gastrointestinal tracts also exhibited variable proliferation of epithelial cells and subjacent fibroblasts. Epithelial intracytoplasmic inclusion bodies, consistent with Guarnieri bodies, were usually inconspicuous by light microscopy, but when present, were most readily apparent in the stratified squamous epithelium of the oral mucosa and epidermis. Multinucleated syncytial cells were also occasionally observed in the stratified squamous epithelium of the tongue, tonsil, and skin, and in the intestinal mucosa. Monkeypox virus antigen was readily demonstrated by immunohistochemistry using anti-vaccinia mouse polyclonal antibodies as well as anti-monkeypox rabbit polyclonal antibodies. Detectable poxviral antigen was limited to sites exhibiting obvious morphologic involvement and was most prominent within epithelial cells, macrophages, dendritic cells, and fibroblasts of affected tissues. The presence of poxviral antigen, as determined by immunohistochemistry, correlated with ultrastructural identification of replicating virus. Concurrent bacterial septicemia, present in one monkey, was associated with increased dissemination of the virus to the liver, spleen, and bone marrow and resulted in a more rapidly fatal clinical course (Zaucha et al., 2001).
  2. Nonhuman primate host:
    1. Taxonomy Information:
      1. Species:
        1. Primates, primates :
          • GenBank Taxonomy No.: 9443
          • Scientific Name: Primata (NCBI Taxonomy)
          • Description: Surveys conducted in areas where human monkeypox cases have occurred have shown 20% (11/55) to 23% (50/215) prevalence of poxvirus neutralizing antibodies in nonhuman primates (Breman et al., 1980).
          • Variant(s):
            • Hominidae :
              • GenBank Taxonomy No.: 9604
              • Scientific Name: Hominidae (NCBI Taxonomy)
              • Common Name: Great apes (NCBI Taxonomy)
              • Description: Out of 39 primates collected during a survey (from a forest), three were found seropositive for monkeypox. At the moment, however, it is difficult to say whether the primates play an important role in sustaining virus transmission in nature, or (like human beings) that they are an occasional host of the virus (Khodakevich et al., 1987).
            • Pan troglodytes :
              • GenBank Taxonomy No.: 9598
              • Scientific Name: Pan troglodytes (NCBI Taxonomy)
              • Common Name: Chimpanzee (NCBI Taxonomy)
              • Description: A case of monkeypox infection in a six-month-old baby girl who had been bitten by a wild chimpanzee in Kivu, Zaire, was investigated. The child had not been exposed to any monkeypox-like disease and no cases of such disease had occurred in the surrounding area during previous months. The time of onset of rash was consistent with the virus having been transmitted from the chimpanzee. However, it is still not known whether chimpanzees and other primates or lower mammals are the primary reservoir of monkeypox infection (Mutombo et al., 1983).
            • Cercopithecidae :
              • GenBank Taxonomy No.: 9527
              • Scientific Name: Cercopithecidae (NCBI Taxonomy)
              • Common Name: Monkey, monkeys, Old World monkeys (Neubauer et al., 1998)
              • Description: Von Magnus described a non-fatal pox disease occurring in Cynomolgus monkeys in Copenhagen in 1958. The etiological agent, named monkeypox, has since been identified in outbreaks of illness in monkeys or apes in the USA, the Netherlands, and France. The strains of monkeypox virus isolated during these outbreaks were found to represent a uniform group differing little from the prototype Copenhagen strain (Lourie et al., 1972).

    2. Infection Process:

      No infection process information is currently available here.

    3. Disease Information:

      No disease information is currently available here.

    4. Prevention:

      No prevention information is currently available here.

    5. Model System:

      No model system information is currently available here.

  3. Non primate host:
    1. Taxonomy Information:
      1. Species:
        1. Animalia, multicellular animals, animals, metazoans :
          • GenBank Taxonomy No.: 33208
          • Scientific Name: Metazoa (NCBI Taxonomy)
          • Description: When the research on the ecology of monkeypox virus entered the latest stage in 1984, three groups of animals were considered priority candidates for maintenance of virus circulations in nature because of their relatively higher population density: terrestrial rodents, squirrels, and gregarious bats (Khodakevich et al., 1987). In one of these studies antibodies were found in rodents, other larger mammals, and birds in the forest areas of Ivory Coast and antibodies have also been found in rodents in Zaire. However, only recently has it been possible to determine that these antibodies had developed specifically in response to monkeypox infection and not to infection caused by any other orthopoxvirus species that might have infected the mammals and birds (Breman et al., 1980).
          • Variant(s):
            • Myrmecophagidae :
              • GenBank Taxonomy No.: 9349
              • Scientific Name: Myrmecophagidae (NCBI Taxonomy)
              • Common Name: Anteaters (NCBI Taxonomy)
              • Description: Monkeypox "Utrecht 65-32" was isolated from a giant anteater during an epizootic of pox-like disease at Rotterdam in 1965 (Lourie et al., 1972).
            • Chiroptera :
              • GenBank Taxonomy No.: 9397
              • Scientific Name: Chiroptera (NCBI Taxonomy)
              • Common Name: gregarious bats (Khodakevich et al., 1987)
              • Description: In 1984, three groups of animals were considered priority candidates for maintenance of virus circulations in nature because of their relatively higher population density: terrestrial rodents, squirrels, and gregarious bats (Khodakevich et al., 1987).
            • Cricetomys emini :
              • GenBank Taxonomy No.: 10084
              • Scientific Name: Cricetomys emini (Hutson et al., 2007)
              • Common Name: Giant pouched rat (Hutson et al., 2007)
              • Description: Among the imported African rodents that were examined, three species showed evidence of MPXV infection. These included three rope squirrels (Funisciuris sp.), two giant pouched rats (Cricetomys spp.), and nine dormice (Graphiuris sp.) (Hutson et al., 2007).
            • Funisciurus :
              • GenBank Taxonomy No.: 226679
              • Scientific Name: Funisciurus (Hutson et al., 2007)
              • Common Name: Rope squirrels (Hutson et al., 2007)
              • Description: MPXV was isolated once from a moribund rope squirrel (Funisciuris anerythrus) found in Zaire (Hutson et al., 2007). Animal antibody surveys in the Democratic Republic of Congo (DRC; former Zaire) suggested that squirrels play a major role as a reservoir of Monkeypox virus and that humans are sporadically infected (Hutin et al., 2001).
            • Graphiuris sp. :
              • GenBank Taxonomy No.: 51345
              • Scientific Name: Graphiuris sp (Hutson et al., 2007).
              • Common Name: Dormice (Hutson et al., 2007)
              • Description: Three species, showed evidence of MPXV infection among imported African rodents. These included rope squirrels (Funisciuris sp.), giant pouched rats (Cricetomys spp.), and dormice (Graphiuris sp.) (Hutson et al., 2007).

    2. Infection Process:

      No infection process information is currently available here.

    3. Disease Information:

      No disease information is currently available here.

    4. Prevention:

      No prevention information is currently available here.

    5. Model System:

      No model system information is currently available here.

  4. Birds:
    1. Taxonomy Information:
      1. Species:
        1. Birds :
          • GenBank Taxonomy No.: 8782
          • Scientific Name: Aves (NCBI Taxonomy)
          • Description: In a survey conducted in areas where human monkeypox cases have occurred antibodies were found in rodents, other larger mammals, and birds in the forest areas of Ivory Coast. However, only recently has it been possible to determine that these antibodies had developed specifically in response to monkeypox infection (Breman et al., 1980).

    2. Infection Process:

      No infection process information is currently available here.

    3. Disease Information:

      No disease information is currently available here.

    4. Prevention:

      No prevention information is currently available here.

    5. Model System:

      No model system information is currently available here.


IV. Labwork Information

A. Biosafety Information:
  1. General biosafety information :
    • Biosafety Level: Level 2 and Level 3 (Jezek and Fenner, 1988).
    • Applicable: Laboratory facilities for handling and storing monkeypox virus should be at biosafety level 2. In addition, all who enter the laboratory must have been vaccinated with smalllpox vaccine within the last three years. Animals infected with monkeypox virus should be kept and handled in a laboratory facility designed for biosafety level 3 (Jezek and Fenner, 1988).
    • Precautions:
      • Once the disease agent is identified, quarantine and immediate ring vaccination are the only effective public health protective procedures, because there is no effective, licensed antiviral therapy for monkeypox. Given the ease of transmission through direct contact and aerosol particles, specimens such as scab or other cutanenous tissues should be handled with care and collected aseptically with respiratory precautions (Nalca et al., 2005).
B. Culturing Information:
  1. Cell culture :
    1. Description: Most orthopox viruses can be grown in one or another kind of cultured cell and assayed by plaque counts in suitable susceptible cells. Species with a restricted host range, such as variola virus, replicate in a narrower range of cells and often produce hyperplastic foci. However, on serial passage, adaptation occurs readily and may involve change to a more lytic plaque. Monolayers infected with viruses that produce hyperplastic foci usually yield much less virus than those infected with viruses that produce lytic plaques, since most cells in the monolayer remain uninfected. Differential growth capacity in particular cell lines (e.g., the rabbit cell line RK 13 and pig embryo kidney cells) may be useful in distinguishing between variola and monkeypox viruses when these viruses are first inoculated into such cells; however, adaptation occurs readily (Fenner et al., 1988(b)).

    2. Medium:
      1. Most human and non-human primate cells, and some cells derived from other species (rabbit kidney and pig embryo kidney cells) are susceptible to infection with variola virus. Growth in pig embryo kidney cells was sometimes used to differentiate between variola and monkeypox viruses (Fenner et al., 1988(b)).
C. Diagnostic Tests :
  1. Organism Detection Tests:
    1. Transmission electron microscopy:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Widespread use of electron microscopy as a diagnostic method was not feasible until the negative staining technique was introduced. Others have shown the value of this method for recognizing poxvirus or herpesvirus particles in vesicle fluid and scabs taken directly from patients (Fenner et al., 1988(b)). Electron microscopy had the advantage of being much the most rapid method of making a presumptive diagnosis, which was a very important requirement, especially in nonendemic countries. In scabs or material that had been some time in transit, it was also the most sensitive, although fields might have to be searched for as long as 30 minutes before a specimen was declared negative (Fenner et al., 1988(a)). Transmission electron microscopy was performed on glutaraldehyde-fixed skin-biopsy specimens from two patients. Virions in various stages of assembly were observed within the cytoplasm of keratinocytes (Panel 3D). Cross sections of mature virions contained dumbbell-shaped cores characteristic of poxviruses (Panel 3E). Negative-stain electron microscopy with phosphotungstic acid was performed on cultures from a patient and Prairie dog and revealed numerous brick-shaped virions with regularly spaced, threadlike ridges on the exposed surfaces (Panel 3F). Taken together, the cell-culture and ultrastructural features suggested that the virus was a member of the genus orthopoxvirus (Reed et al., 2004).
    2. light microscopy:
      1. Time to Perform: 1-to-2-days
      2. Description: The specific tests used to confirm the diagnosis of monkeypox for individual patients varied depending on the availability of specimens and the stage of illness. Sections of formalin-fixed, paraffin-embedded skin-biopsy specimens obtained at the pustular stage from two patients and stained with hematoxylin and eosin showed marked ballooning degeneration of keratinocytes with epidermal necrosis and spongiotic edema (Panel A). In portions of the epidermis, keratinocytes exhibited multinucleation, nuclear clearing with margination of chromatin, and occasional eosinophilic cytoplasmic inclusions (Panel B). A moderate neutrophilic and lymphocytic inflammatory infiltrate was present within the epidermis and superficial dermis (Reed et al., 2004).
    3. Immunohistochemical staining:
      1. Time to Perform: 1-to-2-days
      2. Description: Orthopoxviral antigen was detected in skin-biopsy specimens by means of immunohistochemical staining with rabbit anti-vaccinia polyclonal antibody (Virostat). Antigen was most prominent in degenerating keratinocytes and follicular epithelium but was absent in normal-appearing skin at the edges of the specimen (Panel C). Immunohistochemical stains for herpes simplex viruses 1 and 2 and cytomegalovirus were negative (Reed et al., 2004).

  2. Immunoassay Tests:
    1. Enzyme-Linked Immunosorbent Assay:
      1. Time to Perform: 1-to-2-days
      2. Description: A monkeypox outbreak occurred in the United States in 2003. Serum samples from patients were tested by using an immunoglobulin M (IgM)-capture and an IgG enzyme-linked immunosorbent assay (ELISA) against Orthopoxvirus antigen. The detection of antiviral IgG and IgM antibodies and the kinetics of the antiviral IgG and IgM antibody responses were evaluated. Patients were classified as confirmed, probable, or suspect cases or were excluded as cases based on laboratory test results and epidemiologic and clinical criteria. A total of 37 confirmed case patients with monkeypox were identified, and 116 patients were excluded as case patients based on molecular testing or insufficient epidemiology and clinical data to warrant classification as a suspect or probable case. Of 37 confirmed case patients, 36 had a known history (presence or absence) of smallpox vaccination. Of those, 29 of the 36 either had or developed an IgG response, while 34 of the 36 developed an IgM response, regardless of vaccination status. Serum collected > or =5 days for IgM detection or serum collected > or =8 days after rash onset for IgG detection was most efficient for the detection of monkeypox virus infection. IgM ELISA detects recent infection with orthopoxviruses and, in this case, recent infection with monkeypox virus. In addition, analysis of paired sera for IgG and IgM detected seroconversion, another indicator of recent infection. The ELISA results correlated with the virologic PCR and viral culture results, indicating its diagnostic capabilities for monkeypox (Karem et al., 2005).

  3. Nucleic Acid Detection Tests: :
    1. PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Rapid identification and differentiation of orthopoxviruses by PCR were achieved with primers based on genome sequences encoding the hemagglutinin (HA) protein, an infected-cell membrane antigen that distinguishes orthopoxviruses from other poxvirus genera. The initial identification step used a primer pair of consensus sequences for amplifying an HA DNA fragment from the three known North American orthopoxviruses (raccoonpox, skunkpox, and volepox viruses), and a second pair for amplifying virtually the entire HA open reading frame of the Eurasian-African orthopoxviruses (variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and gerbilpox viruses). RsaI digest electropherograms of the amplified DNAs of the former subgroup provided species differentiation, and TaqI digests differentiated the Eurasian-African orthopoxviruses, including vaccinia virus from the vaccinia virus subspecies buffalopox virus. Endonuclease HhaI digest patterns distinguished smallpox variola major viruses from alastrim variola minor viruses. For the Eurasian-African orthopoxviruses, a confirmatory step that used a set of higher-sequence-homology primers was developed to provide sensitivity to discern individual virus HA DNAs from cross-contaminated orthopoxvirus DNA samples; TaqI and HhaI digestions of the individual amplified HA DNAs confirmed virus identity. Finally, a set of primers and modified PCR conditions were developed on the basis of base sequence differences within the HA genes of the 10 species, which enabled production of a single DNA fragment of a particular size that indicated the specific species (Ropp et al., 1995).
      3. Primers:
        • Consensus sequence primer pairs for amplifying fragments within the HA open reading frame of Eurasian-African orthopoxvirus subgroups: EACP1, EACP2
          • Forward: EACP1: 5' ATG ACA CGA TTG CCA ATA C 3'
          • Reverse: EACP2: 5' CTA GAC TTT GTT TTC TG 3'
          • Product
        • Specific sequence primers for biased amplification of the HA open reading frame of particular Eurasian-African orthopoxviruses. Primer pairs used to amplify selected species e.g. Monkeypox: G-MPV, G-EMVRB
          • Forward: G-MPV: 5' ATG ACA CAA TTA CCA ATA C 3'
          • Reverse: G-EMVRB: 5' CTA GAC TTT GTT CTC TG 3
        • Species-specific primers within the HA open reading frame of orthopoxviruses e.g. Monkeypox
          • Forward: MPV1: 5' CTG ATA ATG TAG AAG AC 3'
          • Reverse: MPV2: 5' TTG TAT TTA CGT GGG TG 3'
          • Product
    2. Real-Time PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Two real-time PCR assays were critical for laboratory diagnosis of monkeypox during the 2003 US outbreak. The E9L-NVAR and B6R assays target orthopoxvirus DNA polymerase and extracellular enveloped protein genes, respectively. These assays are highly sensitive and specific. The E9L-NVAR assay detects 13 Eurasian orthopoxviruses but not variola or North American orthopoxviruses, and the B6R assay detects MPXV isolates but no other orthopoxviruses. Neither assay gave false positives with other rash illness-causing viruses or bacteria. During the 2003 US monkeypox outbreak, the E9L-NVAR and B6R assays provided reliable and sensitive identification of human monkeypox infections within a clinical context. Assay compatibility with multiple real-time PCR platforms allowed simultaneous testing of suspect samples for orthopoxvirus (E9L-NVAR) and MPXV (B6R) DNA. The B6R assay was designed to detect Congo Basin MPXV; West African/US MPXV has one SNP within the B6R probe. The lack of complete homology to the US monkeypox isolates did not adversely affect the detection of MPXV DNA within human samples, confirming the B6R assay diagnostic utility for both known MPXV clades. E9L-NVAR and B6R assays demonstrate 100% specificity for non-variola Eurasian orthopoxvirus and MPXV, respectively. Using two discrete viral gene targets, these assays together provide a reliable and sensitive method for quickly confirming monkeypox infections (Li et al., 2006).
      3. Primers:
        • Generic E9L Orthopoxvirus real-time PCR assay primers
          • Forward: E9L forward primer: 5'-TCA ACT GAA AAG GCC ATC TAT GA-3' (Li et al., 2006)
          • Reverse: E9L reverse primer: 5'-GAG TAT AGA GCA CTA TTT CTA AAT CCC A-3' (Li et al., 2006)
        • B6R MPXV-specific assay primer
          • Forward: B6R forward primer: 5'-ATT GGT CAT TAT TTT TGT CAC AGG AAC A-3' (Li et al., 2006)
          • Reverse: B6R reverse primer: 5'-AAT GGC GTT GAC AAT TAT GGG TG-3' (Li et al., 2006)
    3. PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Orthopoxvirus species were identified and differentiated by polymerase chain reaction amplification of genome DNA using a single primer-pair based on sequences coding for the major protein component of the cowpox virus acidophilic-type inclusion body (ATI). DNA available for 6 of 8 Old World (cowpox, variola, monkeypox, camelpox, ectromelia and vaccinia viruses) and 3 New World (skunkpox, volepox, and raccoonpox) resulted in amplicons that ranged in size from 510 to 1673 base pairs depending on the species, except for raccoonpox virus DNA which did not amplify. XbaI digest gel electrophoresis profiles of the amplicons improved resolution of the differences (Meyer et al., 1997).
      3. Primers:
        • ATI-up-l, ATI-low-l
          • Forward: ATI-up-l: 5'-AATACAAGGAGGATCT-3'
          • Reverse: ATI-low-l: 5'-CTTAACTTTTTCTTTCTC-3'
          • Product
    4. PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: The open reading frame coding for the A-type inclusion body protein (ATI) of monkeypox virus (MPV) was identified and sequenced for two strains. Nucleotide sequence comparison revealed 72-95.3% homology with the reported open reading frame sequences of the ATIs of other orthopoxvirus species, such as variola, vaccinia, cowpox, ectromelia, and camelpox viruses. Each MPV strain contained an 8-bp deletion, which caused a frameshift that introduced a premature stop in the open reading frame at base 2091 relative to the ATI open reading frame of cowpox virus strain Brighton. The sequences enabled a primer pair to be designed that flanked the deletion and specifically amplified a 601-bp fragment that identified and differentiated 19 MPV strains examined from five other Old World orthopoxvirus species examined. The specificity was confirmed by cleavage of the 19 MPV strain amplicons with BglII, which produced three subfragments of expected sized, based on the determined MPV sequences (Neubauer et al., 1998).
      3. Primers:
        • Gabon-1, Gabon-2
          • Forward: Gabon-1: 5'-GAGAGAATCTCTTGATAT-3'
          • Reverse: Gabon-2: 5'-ATTCTAGATTGTAATC-3'
          • Product
    5. PCR-Hybridization:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: A method for species-specific detection of orthopoxviruses pathogenic for humans and animals is described. The method is based on hybridization of a fluorescently labeled amplified DNA specimen with the oligonucleotide DNA probes immobilized on a microchip (MAGIChip). The probes identify species-specific sites within the crmB gene encoding the viral analogue of tumor necrosis factor receptor, one of the most important determinants of pathogenicity in this genus of viruses. The diagnostic procedure takes 6 h and does not require any sophisticated equipment (a portable fluorescence reader can be used) (Lapa et al., 2002).
      3. Primers:
        • TNFR1f, TNFR3r
          • Forward: TNFR1f: 5'-GCT TCC AGA TTA TGT GAT AGC AAG ACT A-3' (Lapa et al., 2002)
          • Reverse: TNFR3r: 5'-TCC GGA TAC TCC GTA TCC TAT TCC-3' (Lapa et al., 2002) A fragment of the vTNFR gene 245 to 273 bp in length (the length varies from one species to another) was amplified using primers TNFR1f and TNFR3r. The primer TNFR3r used in the second round of amplification was 5' fluorescently labeled with Texas Red according to the manufacturer's instructions (Lapa et al., 2002).
    6. Real-Time PCR Assay:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets were designed and extensively tested. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses (Kulesh et al., 2004).
      3. Primers:
        • Monkeypox F3L gene: F3L-F290, F3L-R396
          • Forward: F3L-F290: 5'-CTC ATT GAT TTT TCG CGG GAT A-3'
          • Reverse: F3L-R396: 5'-GAC GAT ACT CCT CCT CGT TGG T-3'
          • Product
        • Monkeypox N3R gene: N3R-F319, N3R-R457
          • Forward: N3R-F319: 5'-AAC AAC CGT CCT ACA ATT AAA CAA CA-3'
          • Reverse: N3R-R457: 5'-CGC TAT CGA ACC ATT TTT GTA GTC T-3'
          • Product

  4. Other Types of Diagnostic Tests:

    No other tests available here.


V. References

A. Journal References:
Berting et al., 2005: Berting A, Goerner W, Spruth M, Kistner O, Kreil TR. Effective poxvirus removal by sterile filtration during manufacture of plasma derivatives. J Med Virol. 2005; 75(4): 603 - 607. [PubMed: 15714487].
Breman et al., 1980: Breman JG, Kalisa-Ruti, Steniowski MV, Zanotto E, Gromyko AI, Arita I. Human monkeypox, 1970-79. Bull World Health Organ. 1980; 58(2): 165 - 182. [PubMed: 6249508].
Centers for Disease Control and Prevention (CDC), 1997: Centers for Disease Control and Prevention (CDC) Human monkeypox--Kasai Oriental, Zaire, 1996-1997. MMWR Morb Mortal Wkly Rep. 1997; 46(14): 304 - 307. [PubMed: 9132583].
Di Giulio and Eckburg, 2004: Di Giulio DB, Eckburg PB. Human monkeypox: an emerging zoonosis. Lancet Infect Dis. 2004; 4(1): 15 - 25. [PubMed: 14720564].
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B. Book References:
Committee on the Assessment of Future Scientific Needs for Variola Virus, Institute of Medicine, 1999: Institute of Medicine (IOM). SMALLPOX AND ITS CONTROL: Variola Virus and Other Orthopoxviruses. 17 - 24. In: Institute of Medicine (IOM). Assessment of Future Scientific Needs for Live Variola Virus 1999. The National Academy Press, Washington, D.C. USA.
Fenner et al., 1988(a): Fenner F., Henderson DA., Arita I., Jezek Z., Ladnyi ID. Chapter 2: Variola virus and other orthopoxviruses. 69 - 119. In: World Health Organization (WHO). Smallpox and its eradication 1988. World Health Organization (WHO), Geneva.
Fenner et al., 1988(b): Fenner F., Henderson DA., Arita I., Jezek Z., Ladnyi ID. Chapter 1: The clinical features of smallpox. 1 - 98. In: World Health Organization (WHO). Smallpox and its eradication 1988. World Health Organization (WHO), Geneva.
Jezek and Fenner, 1988: Jezek Zdenek, Fenner Frank. Human Monkeypox. 1 - 124. In: Melnick Joseph L. Monographs in Virology 1988. Karger, Basel . Munchen (Munich) . Paris . London . New York . New Delhi . Singapore . Tokyo . Sydney.
C. Website References:
NCBI Taxonomy: Monkeypox virus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10244&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Taxonomy: Callithrix jacchus orthopoxvirus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=243803&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Taxonomy: Monkeypox virus (strain Sierra Leone 70-0266) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=130669&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Taxonomy: Monkeypox virus (strain Zaire 77-0666) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=130670&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Entrez: Monkeypox virus, complete genome [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genome&cmd=Retrieve&dopt=Overview&list_uids=15885 ].
NCBI Taxonomy: Myrmecophagidae [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9349&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Taxonomy: Hominidae [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9604&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Taxonomy: Pan troglodytes [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9598&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Taxonomy: Metazoa [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=33208&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Taxonomy: Cercopithecidae [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9527&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Taxonomy: Platyrrhini [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9479&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Taxonomy: Homo sapiens [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Taxonomy: Chiroptera [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9397&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Taxonomy: Aves [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8782&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
NCBI Taxonomy: Primates [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9443&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
D. Thesis References:

No thesis or dissertation references used.


VI. Curation Information