Assessing Monkeypox Virus Prevalence in Small Mammals at the Human-Animal Interface in the Democratic Republic of the Congo.

During 2012, 2013 and 2015, we collected small mammals within 25 km of the town of Boende in Tshuapa Province, the Democratic Republic of the Congo. The prevalence of monkeypox virus (MPXV) in this area is unknown; however, cases of human infection were previously confirmed near these collection sites. Samples were collected from 353 mammals (rodents, shrews, pangolins, elephant shrews, a potamogale, and a hyrax). Some rodents and shrews were captured from houses where human monkeypox cases have recently been identified, but most were trapped in forests and agricultural areas near villages. Real-time PCR and ELISA were used to assess evidence of MPXV infection and other Orthopoxvirus (OPXV) infections in these small mammals. Seven (2.0%) of these animal samples were found to be anti-orthopoxvirus immunoglobulin G (IgG) antibody positive (six rodents: two Funisciurus spp.; one Graphiurus lorraineus; one Cricetomys emini; one Heliosciurus sp.; one Oenomys hypoxanthus, and one elephant shrew Petrodromus tetradactylus); no individuals were found positive in PCR-based assays. These results suggest that a variety of animals can be infected with OPXVs, and that epidemiology studies and educational campaigns should focus on animals that people are regularly contacting, including larger rodents used as protein sources.

Laboratory Investigations of African Pouched Rats (Cricetomys gambianus) as a Potential Reservoir Host Species for Monkeypox Virus.

Monkeypox is a zoonotic disease endemic to central and western Africa, where it is a major public health concern. Although Monkeypox virus (MPXV) and monkeypox disease in humans have been well characterized, little is known about its natural history, or its maintenance in animal populations of sylvatic reservoir(s). In 2003, several species of rodents imported from Ghana were involved in a monkeypox outbreak in the United States with individuals of three African rodent genera (Cricetomys, Graphiurus, Funisciurus) shown to be infected with MPXV. Here, we examine the course of MPXV infection in Cricetomys gambianus (pouched Gambian rats) and this rodent species' competence as a host for the virus. We obtained ten Gambian rats from an introduced colony in Grassy Key, Florida and infected eight of these via scarification with a challenge dose of 4X104 plaque forming units (pfu) from either of the two primary clades of MPXV: Congo Basin (C-MPXV: n = 4) or West African (W-MPXV: n = 4); an additional 2 animals served as PBS controls. Viral shedding and the effect of infection on activity and physiological aspects of the animals were measured. MPXV challenged animals had significantly higher core body temperatures, reduced activity and increased weight loss than PBS controls. Viable virus was found in samples taken from animals in both experimental groups (C-MPXV and W-MPXV) between 3 and 27 days post infection (p.i.) (up to 1X108 pfu/ml), with viral DNA found until day 56 p.i. The results from this work show that Cricetomys gambianus (and by inference, probably the closely related species, Cricetomys emini) can be infected with MPXV and shed viable virus particles; thus suggesting that these animals may be involved in the maintenance of MPXV in wildlife mammalian populations. More research is needed to elucidate the epidemiology of MPXV and the role of Gambian rats and other species.

Zoonotic parapoxviruses detected in symptomatic cattle in Bangladesh.

BACKGROUND: Application of molecular diagnostic methods to the determination of etiology in suspected poxvirus-associated infections of bovines is important both for the diagnosis of the individual case and to form a more complete understanding of patterns of strain occurrence and spread. The objective of this study was to identify and characterize bovine-associated zoonotic poxviruses in Bangladesh which are relevant to animal and human health. FINDINGS: Investigators from the International Center Diarrhoeal Disease Research (icddr,b), the US Centers for Disease Control and Prevention (CDC), and the Bangladesh Department of Livestock Services traveled to three districts in Bangladesh-Siranjganj, Rangpur and Bhola-to collect diagnostic specimens from dairy cattle and buffalo that had symptoms consistent with poxvirus-associated infections. Bovine papular stomatitis virus (BPSV) DNA was obtained from lesion material (teat) and an oral swab collected from an adult cow and calf (respectively) from a dairy production farm in Siranjganj. Pseudocowpox virus (PCPV) DNA signatures were obtained from a scab and oral swab collected from a second dairy cow and her calf from Rangpur. CONCLUSIONS: We report the first detection of zoonotic poxviruses from Bangladesh and show phylogenetic comparisons between the Bangladesh viruses and reference strains based on analyses of the B2L and J6R loci (vaccinia orthologs). Understanding the range and diversity of different species and strains of parapoxvirus will help to spotlight unusual patterns of occurrence that could signal events of significance to the agricultural and public health sectors.

A new hero emerges: another exceptional mammalian spine and its potential adaptive significance.

The hero shrew's (Scutisorex somereni) massive interlocking lumbar vertebrae represent the most extreme modification of the vertebral column known in mammals. No intermediate form of this remarkable morphology is known, nor is there any convincing theory to explain its functional significance. We document a new species in the heretofore monotypic genus Scutisorex; the new species possesses cranial and vertebral features representing intermediate character states between S. somereni and other shrews. Phylogenetic analyses of DNA sequences support a sister relationship between the new species and S. somereni. While the function of the unusual spine in Scutisorex is unknown, it gives these small animals incredible vertebral strength. Based on field observations, we hypothesize that the unique vertebral column is an adaptation allowing these shrews to lever heavy or compressive objects to access concentrated food resources inaccessible to other animals.

Elucidating the role of the complement control protein in monkeypox pathogenicity.

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

Vaccinia virus infections in martial arts gym, Maryland, USA, 2008.

Vaccinia virus is an orthopoxvirus used in the live vaccine against smallpox. Vaccinia virus infections can be transmissible and can cause severe complications in those with weakened immune systems. We report on a cluster of 4 cases of vaccinia virus infection in Maryland, USA, likely acquired at a martial arts gym.

Dosage comparison of Congo Basin and West African strains of monkeypox virus using a prairie dog animal model of systemic orthopoxvirus disease.

The prairie dog is valuable for the study of monkeypox virus (MPXV) virulence and closely resembles human systemic orthopoxvirus disease. Herein, we utilize a variable dose intranasal challenge with approximately 10(3), 10(4), 10(5), and 10(6)PFU for each clade to further characterize virulence differences between the two MPXV clades. A trend of increased morbidity and mortality as well as greater viral shedding was observed with increasing viral challenge dose. Additionally, there appeared to be a delay in onset of disease for animals challenged with lower dosages of virus. Mathematical calculations were used to determine LD(50) values and based on these calculations, Congo Basin MPXV had approximately a hundred times lower LD(50) value than the West African clade (5.9x10(3) and 1.29x10(5) respectively); reinforcing previous findings that Congo Basin MPXV is more virulent.

Comparison of West African and Congo Basin monkeypox viruses in BALB/c and C57BL/6 mice.

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

A prairie dog animal model of systemic orthopoxvirus disease using West African and Congo Basin strains of monkeypox virus.

Multiple monkeypox virus (MPXV) animal models have been discussed in previous studies, but no small animal models, nor most non-human primate models, demonstrated the protracted asymptomatic incubation phase seen in systemic human orthopoxvirus illness. Herein, we characterize a black-tailed prairie dog (PD) (Cynomys ludovicianus) model of infection, via intranasal and intradermal exposures, with the two MPXV clades. Daily observations of the animals were made (food consumption, general symptoms, disease presentation), while weights and virus evaluations (ocular, nasal, oropharyngeal, faeces, blood) were obtained/made every third day. Generalized rash became apparent 9-12 days post-infection for all animals. Individual animals demonstrated a range of symptoms consistent with human monkeypox disease. Measurable viraemias and excretas were similar for both clade-representative strains and persisted until at least day 21. Greater morbidity was observed in Congo Basin strain-challenged animals and mortality was observed only in the Congo Basin strain-challenged animals. The PD model is valuable for the study of strain-dependent differences in MPXV. Additionally, the model closely mimics human systemic orthopoxvirus disease and may serve as a valuable non-human surrogate for investigations of antivirals and next generation orthopoxvirus vaccines.

Monkeypox zoonotic associations: insights from laboratory evaluation of animals associated with the multi-state US outbreak.

At the onset of the 2003 US monkeypox outbreak, virologic data were unavailable regarding which animal species were involved with virus importation and/or subsequent transmission to humans and whether there was a risk for establishment of zoonotic monkeypox in North America. Similarly, it was unclear which specimens would be best for virus testing. Monkeypox DNA was detected in at least 33 animals, and virus was cultured from 22. Virus-positive animals included three African species associated with the importation event (giant pouched rats, Cricetomys spp.; rope squirrels, Funisciuris sp.; and dormice, Graphiuris sp.). 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.

Prevalence of antibodies against orthopoxviruses among residents of Likouala region, Republic of Congo: evidence for monkeypox virus exposure.

Monkeypox virus is a zoonotic orthopoxvirus (OPX) of west and central sub-Saharan Africa. We conducted a cross-sectional serosurvey in Likouala region, Republic of Congo to assess exposure to OPX. Whole blood was collected using Nobuto blood filter strips (NBFS). Titers of IgM and IgG to OPX were assessed using an enzyme-linked immunosorbent assay. Demographic and clinical characteristics were compared with serostatus using the chi-square test or Fisher's exact test. Multivariate logistic regression was performed to evaluate factors for independent association with serostatus. A total of 994 specimens were analyzed; the overall seroprevalence for OPX IgM was 1.7%. Age < 25 years reduced the likelihood of OPX exposure, and persons living in Ngangania village had independently higher odds (odds ratio = 33.5, 95% confidence interval = 7.2-166). Blood collection for serosurveys using NBFS is feasible and practical. Adult activities such as hunting and carcass preparation may play an important role in exposure to Monkeypox virus.

Spectrum of infection and risk factors for human monkeypox, United States, 2003.

For the 2003 monkeypox virus (MPXV) outbreak in the United States, interhuman transmission was not documented and all case-patients were near or handled MPXV-infected prairie dogs. We initiated a case-control study to evaluate risk factors for animal-to-human MPXV transmission. Participants completed a questionnaire requesting exposure, clinical, and demographic information. Serum samples were obtained for analysis of immunoglobulin G (IgG) and IgM to orthopoxvirus. When data were adjusted for smallpox vaccination, case-patients were more likely than controls to have had daily exposure to a sick animal (odds ratio [OR] 4.0, 95% confidence interval [CI] 1.2-13.4), cleaned cages and bedding of a sick animal (OR 5.3, 95% CI 1.4-20.7), or touched a sick animal (OR 4.0, 95% CI 1.2-13.4). These findings demonstrate that human MPXV infection is associated with handling of MPXV-infected animals and suggest that exposure to excretions and secretions of infected animals can result in infection.

Transmission of monkeypox among persons exposed to infected prairie dogs in Indiana in 2003.

OBJECTIVE: To describe a cluster of human monkeypox cases associated with exposure to ill prairie dogs in a home child care. DESIGN, SETTING, PARTICIPANTS: We identified all persons exposed to 2 pet prairie dogs in County A, Indiana; performed active surveillance for symptomatic monkeypox infection; and evaluated the types of exposure that may have resulted in infection. For children who attended the child care where the animals were housed, we also measured the rate of seroconversion to monkeypox virus. MAIN OUTCOME MEASURES: Nine (13%) of 70 persons exposed to the prairie dogs reported signs and symptoms of monkeypox. Two (40%) of 5 symptomatic child care attendees reported direct contact with the prairie dogs. Two (13%) of 15 child care attendees evaluated tested positive for IgM antibodies against orthopoxvirus; both reported symptoms consistent with monkeypox. RESULTS: The risk of symptomatic infection correlated with the time and intensity of animal exposure, which was 100% (4/4) among family members with extensive direct contact, 19% (5/26) among the veterinarian and nonfamily child care attendees with moderate exposure, and 0% (0/40) among school children with limited exposure (P<.01). CONCLUSIONS: Monkeypox virus was transmitted from ill prairie dogs in a child care and veterinary facilities. The risk of symptomatic infection correlated with the amount of exposure to the prairie dogs. Although most cases of human monkeypox were associated with direct animal contact, other routes of transmission cannot be excluded.

Evaluation of human-to-human transmission of monkeypox from infected patients to health care workers.

BACKGROUND: In 2003, human monkeypox was first identified in the United States. The outbreak was associated with exposure to infected prairie dogs, but the potential for person-to-person transmission was a concern. This study examines health care worker (HCW) exposure to 3 patients with confirmed monkeypox. METHODS: Exposed HCWs, defined as HCWs who entered a 2-m radius surrounding case patients with confirmed monkeypox, were identified by infection-control practitioners. A self-administered questionnaire and analysis of paired serum specimens determined exposure status, immune response, and postexposure signs and symptoms of monkeypox. RESULTS: Of 81 exposed HCWs, 57 (70%) participated in the study. Among 57 participants, 40 (70%) had > or =1 unprotected exposure; none reported signs or symptoms consistent with monkeypox illness. One exposed HCW (2%), who had been vaccinated for smallpox within the past year, had serological evidence of recent orthopoxvirus infection; acute- and convalescent-phase serum specimens tested positive for anti-orthopoxvirus IgM. No exposed HCWs had signs and symptoms consistent with monkeypox. CONCLUSION: More than three-quarters of exposed HCWs reported at least 1 unprotected encounter with a patient who had monkeypox. One asymptomatic HCW showed laboratory evidence of recent orthopoxvirus infection, which was possibly attributable to either recent infection or smallpox vaccination. Transmission of monkeypox likely is a rare event in the health care setting.