Last data update: Nov 04, 2024. (Total: 48056 publications since 2009)
Records 1-30 (of 31 Records) |
Query Trace: Karem KL[original query] |
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Design and optimization of a monkeypox virus specific serological assay
Taha TY , Townsend MB , Pohl J , Karem KL , Damon IK , Mbala Kingebeni P , Muyembe Tamfum JJ , Martin JW , Pittman PR , Huggins JW , Satheshkumar PS , Bagarozzi DA Jr , Reynolds MG , Hughes LJ . Pathogens 2023 12 (3) Monkeypox virus (MPXV), a member of the Orthopoxvirus (OPXV) genus, is a zoonotic virus, endemic to central and western Africa that can cause smallpox-like symptoms in humans with fatal outcomes in up to 15% of patients. The incidence of MPXV infections in the Democratic Republic of the Congo, where the majority of cases have occurred historically, has been estimated to have increased as much as 20-fold since the end of smallpox vaccination in 1980. Considering the risk global travel carries for future disease outbreaks, accurate epidemiological surveillance of MPXV is warranted as demonstrated by the recent Mpox outbreak, where the majority of cases were occurring in non-endemic areas. Serological differentiation between childhood vaccination and recent infection with MPXV or other OPXVs is difficult due to the high level of conservation within OPXV proteins. Here, a peptide-based serological assay was developed to specifically detect exposure to MPXV. A comparative analysis of immunogenic proteins across human OPXVs identified a large subset of proteins that could potentially be specifically recognized in response to a MPXV infection. Peptides were chosen based upon MPXV sequence specificity and predicted immunogenicity. Peptides individually and combined were screened in an ELISA against serum from well-characterized Mpox outbreaks, vaccinee sera, and smallpox sera collected prior to eradication. One peptide combination was successful with ~86% sensitivity and ~90% specificity. The performance of the assay was assessed against the OPXV IgG ELISA in the context of a serosurvey by retrospectively screening a set of serum specimens from the region in Ghana believed to have harbored the MPXV-infected rodents involved in the 2003 United States outbreak. |
IMVAMUNE and ACAM2000 provide different protection against disease when administered postexposure in an intranasal monkeypox challenge prairie dog model
Keckler MS , Salzer JS , Patel N , Townsend MB , Nakazawa YJ , Doty JB , Gallardo-Romero NF , Satheshkumar PS , Carroll DS , Karem KL , Damon IK . Vaccines (Basel) 2020 8 (3) The protection provided by smallpox vaccines when used after exposure to Orthopoxviruses is poorly understood. Postexposu re administration of 1st generation smallpox vaccines was effective during eradication. However, historical epidemiological reports and animal studies on postexposure vaccination are difficult to extrapolate to today's populations, and 2nd and 3rd generation vaccines, developed after eradication, have not been widely tested in postexposure vaccination scenarios. In addition to concerns about preparedness for a potential malevolent reintroduction of variola virus, humans are becoming increasingly exposed to naturally occurring zoonotic orthopoxviruses and, following these exposures, disease severity is worse in individuals who never received smallpox vaccination. This study investigated whether postexposure vaccination of prairie dogs with 2nd and 3rd generation smallpox vaccines was protective against monkeypox disease in four exposure scenarios. We infected animals with monkeypox virus at doses of 10(4) pfu (2× LD(50)) or 10(6) pfu (170× LD(50)) and vaccinated the animals with IMVAMUNE(®) or ACAM2000(®) either 1 or 3 days after challenge. Our results indicated that postexposure vaccination protected the animals to some degree from the 2× LD(50), but not the 170× LD(5) challenge. In the 2× LD(50) challenge, we also observed that administration of vaccine at 1 day was more effective than administration at 3 days postexposure for IMVAMUNE(®), but ACAM2000(®) was similarly effective at either postexposure vaccination time-point. The effects of postexposure vaccination and correlations with survival of total and neutralizing antibody responses, protein targets, take formation, weight loss, rash burden, and viral DNA are also presented. |
Magnitude and diversity of immune response to vaccinia virus is dependent on route of administration
Hughes LJ , Townsend MB , Gallardo-Romero N , Hutson CL , Patel N , Doty JB , Salzer JS , Damon IK , Carroll DS , Satheshkumar PS , Karem KL . Virology 2020 544 55-63 Historic observations suggest that survivors of smallpox maintained lifelong immunity and protection to subsequent infection compared to vaccinated individuals. Although protective immunity by vaccination using a related virus (vaccinia virus (VACV) strains) was the key for smallpox eradication, it does not uniformly provide long term, or lifelong protective immunity (Heiner et al., 1971). To determine differences in humoral immune responses, mice were inoculated with VACV either systemically, using intranasal inoculation (IN), or locally by an intradermal (ID) route. We hypothesized that sub-lethal IN infections may mimic systemic or naturally occurring infection and lead to an immunodominance reaction, in contrast to localized ID immunization. The results demonstrated systemic immunization through an IN route led to enhanced adaptive immunity to VACV-expressed protein targets both in magnitude and in diversity when compared to an ID route using a VACV protein microarray. In addition, cytokine responses, assessed using a Luminex(R) mouse cytokine multiplex kit, following IN infection was greater than that stemming from ID infection. Overall, the results suggest that the route of immunization (or infection) influences antibody responses. The greater magnitude and diversity of response in systemic infection provides indirect evidence for anecdotal observations made during the smallpox era that survivors maintain lifelong protection. These findings also suggest that systemic or disseminated host immune induction may result in a superior response, that may influence the magnitude of, as well as duration of protective responses. |
Assessing monkeypox virus prevalence in small mammals at the human-animal interface in the Democratic Republic of the Congo
Doty JB , Malekani JM , Kalemba LN , Stanley WT , Monroe BP , Nakazawa YU , Mauldin MR , Bakambana TL , Liyandja Dja Liyandja T , Braden ZH , Wallace RM , Malekani DV , McCollum AM , Gallardo-Romero N , Kondas A , Peterson AT , Osorio JE , Rocke TE , Karem KL , Emerson GL , Carroll DS . Viruses 2017 9 (10) 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
Hutson CL , Nakazawa YJ , Self J , Olson VA , Regnery RL , Braden Z , Weiss S , Malekani J , Jackson E , Tate M , Karem KL , Rocke TE , Osorio JE , Damon IK , Carroll DS . PLoS Negl Trop Dis 2015 9 (10) e0004013 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. |
A phylogeographic investigation of African monkeypox.
Nakazawa Y , Mauldin MR , Emerson GL , Reynolds MG , Lash RR , Gao J , Zhao H , Li Y , Muyembe JJ , Kingebeni PM , Wemakoy O , Malekani J , Karem KL , Damon IK , Carroll DS . Viruses 2015 7 (4) 2168-84 Monkeypox is a zoonotic disease caused by a virus member of the genus Orthopoxvirus and is endemic to Central and Western African countries. Previous work has identified two geographically disjuct clades of monkeypox virus based on the analysis of a few genomes coupled with epidemiological and clinical analyses; however, environmental and geographic causes of this differentiation have not been explored. Here, we expand previous phylogenetic studies by analyzing a larger set of monkeypox virus genomes originating throughout Sub-Saharan Africa to identify possible biogeographic barriers associated with genetic differentiation; and projected ecological niche models onto environmental conditions at three periods in the past to explore the potential role of climate oscillations in the evolution of the two primary clades. Analyses supported the separation of the Congo Basin and West Africa clades; the Congo Basin clade shows much shorter branches, which likely indicate a more recent diversification of isolates within this clade. The area between the Sanaga and Cross Rivers divides the two clades and the Dahomey Gap seems to have also served as a barrier within the West African clade. Contraction of areas with suitable environments for monkeypox virus during the Last Glacial Maximum, suggests that the Congo Basin clade of monkeypox virus experienced a severe bottleneck and has since expanded its geographic range. |
Identification of Giardia duodenalis and Enterocytozoon bieneusi in an epizoological investigation of a laboratory colony of prairie dogs, Cynomys ludovicianus.
Roellig DM , Salzer JS , Carroll DS , Ritter JM , Drew C , Gallardo-Romero N , Keckler MS , Langham G , Hutson CL , Karem KL , Gillespie TR , Visvesvara GS , Metcalfe MG , Damon IK , Xiao L . Vet Parasitol 2015 210 91-7 Since 2005, black-tailed prairie dogs (Cynomys ludovicianus) have been collected for use as research animals from field sites in Kansas, Colorado, and Texas. In January of 2012, Giardia trophozoites were identified by histology, thin-section electron microscopy, and immunofluorescent staining in the lumen of the small intestine and colon of a prairie dog euthanized because of extreme weight loss. With giardiasis suspected as the cause of weight loss, a survey of Giardia duodenalis in the laboratory colony of prairie dogs was initiated. Direct immunofluorescent testing of feces revealed active shedding of Giardia cysts in 40% (n=60) of animals held in the vivarium. All tested fecal samples (n=29) from animals in another holding facility where the index case originated were PCR positive for G. duodenalis with assemblages A and B identified from sequencing triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh), and beta-giardin (bg) genes. Both assemblages are considered zoonotic, thus the parasites in prairie dogs are potential human pathogens and indicate prairie dogs as a possible wildlife reservoir or the victims of pathogen spill-over. Molecular testing for other protozoan gastrointestinal parasites revealed no Cryptosporidium infections but identified a host-adapted Enterocytozoon bieneusi genotype group. |
A highly specific monoclonal antibody against monkeypox virus detects the heparin binding domain of A27
Hughes LJ , Goldstein J , Pohl J , Hooper JW , Lee Pitts R , Townsend MB , Bagarozzi D , Damon IK , Karem KL . Virology 2014 464-465c 264-273 The eradication of smallpox and the cessation of global vaccination led to the increased prevalence of human infections in Central Africa. Serologic and protein-based diagnostic assay for MPXV detection is difficult due to cross-reactive antibodies that do not differentiate between diverse orthopoxvirus (OPXV) species. A previously characterized monoclonal antibody (mAb 69-126-3-7) against MPXV [1] was retested for cross-reactivity with various OPXVs. The 14.5kDa band protein that reacted with mAb 69-126-3 was identified to be MPXV A29 protein (homolog of vaccinia virus Copenhagen A27). Amino acid sequence analysis of the MPXV A29 with other OPXV homologs identified four amino acid changes. Peptides corresponding to these regions were designed and evaluated for binding to mAb 69-126-3 by ELISA and BioLayer Interferometry (BLI). Further refinement and truncations mapped the specificity of this antibody to a single amino acid difference in a 30-mer peptide compared to other OPXV homologs. This particular residue is proposed to be essential for heparin binding by VACV A27 protein. Despite this substitution, MPXV A29 bound to heparin with similar affinity to that of VACV A27 protein, suggesting flexibility of this motif for heparin binding. Although binding of mAb 69-126-3-7 to MPXV A29 prevented interaction with heparin, it did not have any effect on the infectivity of MPXV. Characterization of 69-126-3-7 mAb antibody allows for the possibility of the generation of a serological based species-specific detection of OPXVs despite high proteomic homology. |
Poxvirus viability and signatures in historical relics
McCollum AM , Li Y , Wilkins K , Karem KL , Davidson WB , Paddock CD , Reynolds MG , Damon IK . Emerg Infect Dis 2014 20 (2) 177-84 Although it has been >30 years since the eradication of smallpox, the unearthing of well-preserved tissue material in which the virus may reside has called into question the viability of variola virus decades or centuries after its original occurrence. Experimental data to address the long-term stability and viability of the virus are limited. There are several instances of well-preserved corpses and tissues that have been examined for poxvirus viability and viral DNA. These historical specimens cause concern for potential exposures, and each situation should be approached cautiously and independently with the available information. Nevertheless, these specimens provide information on the history of a major disease and vaccination against it. |
Mapping monkeypox transmission risk through time and space in the Congo basin
Nakazawa Y , Lash RR , Carroll DS , Damon IK , Karem KL , Reynolds MG , Osorio JE , Rocke TE , Malekani JM , Muyembe JJ , Formenty P , Peterson AT . PLoS One 2013 8 (9) e74816 Monkeypox is a major public health concern in the Congo Basin area, with changing patterns of human case occurrences reported in recent years. Whether this trend results from better surveillance and detection methods, reduced proportions of vaccinated vs. non-vaccinated human populations, or changing environmental conditions remains unclear. Our objective is to examine potential correlations between environment and transmission of monkeypox events in the Congo Basin. We created ecological niche models based on human cases reported in the Congo Basin by the World Health Organization at the end of the smallpox eradication campaign, in relation to remotely-sensed Normalized Difference Vegetation Index datasets from the same time period. These models predicted independent spatial subsets of monkeypox occurrences with high confidence; models were then projected onto parallel environmental datasets for the 2000s to create present-day monkeypox suitability maps. Recent trends in human monkeypox infection are associated with broad environmental changes across the Congo Basin. Our results demonstrate that ecological niche models provide useful tools for identification of areas suitable for transmission, even for poorly-known diseases like monkeypox. |
Novel poxvirus in big brown bats, northwestern United States
Emerson GL , Nordhausen R , Garner MM , Huckabee JR , Johnson S , Wohrle RD , Davidson WB , Wilkins K , Li Y , Doty JB , Gallardo-Romero NF , Metcalfe MG , Karem KL , Damon IK , Carroll DS . Emerg Infect Dis 2013 19 (6) 1002-4 A wildlife hospital and rehabilitation center in northwestern United States received several big brown bats with necrosuppurative osteomyelitis in multiple joints. Wing and joint tissues were positive by PCR for poxvirus. Thin-section electron microscopy showed poxvirus particles within A-type inclusions. Phylogenetic comparison supports establishment of a new genus of Poxviridae. |
Orthopoxvirus variola infection of Cynomys ludovicianus (North American Black tailed prairie dog)
Carroll DS , Olson VA , Smith SK , Braden ZH , Patel N , Abel J , Li Y , Damon IK , Karem KL . Virology 2013 443 (2) 358-62 Since the eradication of Smallpox, researchers have attempted to study Orthopoxvirus pathogenesis and immunity in animal models in order to correlate results human smallpox. A solely human pathogen, Orthopoxvirus variola fails to produce authentic smallpox illness in any other animal species tested to date. In 2003, an outbreak in the USA of Orthopoxvirus monkeypox, revealed the susceptibility of the North American black-tailed prairie dog (Cynomys ludovicianus) to infection and fulminate disease. Prairie dogs infected with Orthopoxvirus monkeypox present with a clinical scenario similar to ordinary smallpox, including prodrome, rash, and high mortality. This study examines if Black-tailed prairie dogs can become infected with O. variola and serve as a surrogate model for the study of human smallpox disease. Substantive evidence of infection is found in immunological seroconversion of animals to either intranasal or intradermal challenges with O. variola, but in the absence of overt illness. |
Unintentional transfer of vaccinia virus associated with smallpox vaccines: ACAM2000 (R) compared with Dryvax (R)
Tack DM , Karem KL , Montgomery JR , Collins L , Bryant-Genevier MG , Tiernan R , Cano M , Lewis P , Engler RJ , Damon IK , Reynolds MG . Hum Vaccin Immunother 2013 9 (7) 1489-96 BACKGROUND: Routine vaccination against smallpox (variola) ceased in the US in 1976. However, in 2002 limited coverage for military personnel and some healthcare workers was reinstituted. In March 2008, ACAM2000(R) replaced Dryvax(R) as the vaccine used in the United States against smallpox. Unintentional transfer of vaccinia virus from a vaccination site by autoinoculation or contact transmission, can have significant public health implications. We summarize unintentional virus transfer AEs associated with ACAM2000(R) since March 2008 and compare with Dryvax(R). RESULTS: We identified 309 reports for ACAM2000(R) with skin or ocular involvement, of which 93 were autoinoculation cases and 20 were contact transmission cases. The rate for reported cases of autoinoculation was 20.6 per 100,000 vaccinations and for contact transmission was 4.4 per 100,000 vaccinations. Eighteen contact transmission cases could be attributed to contact during a sporting activity (45%) or intimate contact (45%). Of the 113 unintentional transfer cases, 6 met the case definition for ocular vaccinia. The most common locations for all autoinoculation and contact cases were arm/elbow/shoulder (35/113; 31%) and face (24/113; 21%). METHODS: We reviewed 753 reports associated with smallpox in the Vaccine Adverse Event Reporting System and CDC Poxvirus consultation log, reported from March 2008 to August 2010. Reports were classified into categories based upon standard case definitions. CONCLUSION: Overall, unintentional transfer events for ACAM2000(R) and Dryvax(R) are similar. We recommend continued efforts to prevent transfer events and continuing education for healthcare providers focused on recognition of vaccinia lesions, proper sample collection, and laboratory testing to confirm diagnosis. |
Serologic evidence for circulating orthopoxviruses in peridomestic rodents from rural Uganda
Salzer JS , Carroll DS , Rwego IB , Li Y , Falendysz EA , Shisler JL , Karem KL , Damon IK , Gillespie TR . J Wildl Dis 2013 49 (1) 125-31 The prevalence of orthopoxviruses (OPXV) among wildlife, including monkeypox virus (MPXV), remains largely unknown. Outbreaks of human monkeypox in central Africa have been associated with hunting, butchering, and consuming infected forest animals, primarily rodents and primates. Monkeypox cases have not been reported in east Africa, where human contact with wildlife is more limited. Whether this lack of human disease is due to the absence of MPXV in rodents is unknown. However, testing of wildlife beyond the known geographic distribution of human cases of monkeypox has rarely been conducted, limiting our knowledge of the natural distribution of MPXV and other OPXV. To improve our understanding of the natural distribution of OPXV in Africa and related risks to public health, we conducted a serosurvey of peridomestic rodents (Rattus rattus) in and around traditional dwellings in Kabarole District, Uganda, from May 2008 to July 2008. We tested for OPXV antibody in areas free of human monkeypox. Sera from 41% of the R. rattus individuals sampled reacted to OPXV-specific proteins from multiple, purified OPXV samples, but did not react by enzyme-linked immunosorbent assay. The specific OPXV could not be identified because poxvirus DNA was undetectable in corresponding tissues. We conclude that an OPXV or a similar poxvirus is circulating among wild rodents in Uganda. With the known geographic range of OPXV in rodents now increased, factors that dictate OPXV prevalence and disease will be identified. |
Transmissibility of the monkeypox virus clades via respiratory transmission: investigation using the prairie dog-monkeypox virus challenge system
Hutson CL , Gallardo-Romero N , Carroll DS , Clemmons C , Salzer JS , Nagy T , Hughes CM , Olson VA , Karem KL , Damon IK . PLoS One 2013 8 (2) e55488 Monkeypox virus (MPXV) is endemic within Africa where it sporadically is reported to cause outbreaks of human disease. In 2003, an outbreak of human MPXV occurred in the US after the importation of infected African rodents. Since the eradication of smallpox (caused by an orthopoxvirus (OPXV) related to MPXV) and cessation of routine smallpox vaccination (with the live OPXV vaccinia), there is an increasing population of people susceptible to OPXV diseases. Previous studies have shown that the prairie dog MPXV model is a functional animal model for the study of systemic human OPXV illness. Studies with this model have demonstrated that infected animals are able to transmit the virus to naive animals through multiple routes of exposure causing subsequent infection, but were not able to prove that infected animals could transmit the virus exclusively via the respiratory route. Herein we used the model system to evaluate the hypothesis that the Congo Basin clade of MPXV is more easily transmitted, via respiratory route, than the West African clade. Using a small number of test animals, we show that transmission of viruses from each of the MPXV clade was minimal via respiratory transmission. However, transmissibility of the Congo Basin clade was slightly greater than West African MXPV clade (16.7% and 0% respectively). Based on these findings, respiratory transmission appears to be less efficient than those of previous studies assessing contact as a mechanism of transmission within the prairie dog MPXV animal model. |
Detection of human monkeypox in the Republic of the Congo following intensive community education
Reynolds MG , Emerson GL , Pukuta E , Karhemere S , Muyembe JJ , Bikindou A , McCollum AM , Moses C , Wilkins K , Zhao H , Damon IK , Karem KL , Li Y , Carroll DS , Mombouli JV . Am J Trop Med Hyg 2013 88 (5) 982-985 Monkeypox is an acute viral infection with a clinical course resembling smallpox. It is endemic in northern and central Democratic Republic of the Congo (DRC), but it is reported only sporadically in neighboring Republic of the Congo (ROC). In October 2009, interethnic violence in northwestern DRC precipitated the movement of refugees across the Ubangi River into ROC. The influx of refugees into ROC heightened concerns about monkeypox in the area, because of the possibility that the virus could be imported, or that incidence could increase caused by food insecurity and over reliance on bush meat. As part of a broad-based campaign to improve health standards in refugee settlement areas, the United Nations International Children's Emergency Fund (UNICEF) sponsored a program of intensive community education that included modules on monkeypox recognition and prevention. In the 6 months immediately following the outreach, 10 suspected cases of monkeypox were reported to health authorities. Laboratory testing confirmed monkeypox virus infection in two individuals, one of whom was part of a cluster of four suspected cases identified retrospectively. Anecdotes collected at the time of case reporting suggest that the outreach campaign contributed to detection of suspected cases of monkeypox. |
Phylogenetic and ecologic perspectives of a monkeypox outbreak, southern Sudan, 2005.
Nakazawa Y , Emerson GL , Carroll DS , Zhao H , Li Y , Reynolds MG , Karem KL , Olson VA , Lash RR , Davidson WB , Smith SK , Levine RS , Regnery RL , Sammons SA , Frace MA , Mutasim EM , Karsani ME , Muntasir MO , Babiker AA , Opoka L , Chowdhary V , Damon IK . Emerg Infect Dis 2013 19 (2) 237-45 Identification of human monkeypox cases during 2005 in southern Sudan (now South Sudan) raised several questions about the natural history of monkeypox virus (MPXV) in Africa. The outbreak area, characterized by seasonally dry riverine grasslands, is not identified as environmentally suitable for MPXV transmission. We examined possible origins of this outbreak by performing phylogenetic analysis of genome sequences of MPXV isolates from the outbreak in Sudan and from differing localities. We also compared the environmental suitability of study localities for monkeypox transmission. Phylogenetically, the viruses isolated from Sudan outbreak specimens belong to a clade identified in the Congo Basin. This finding, added to the political instability of the area during the time of the outbreak, supports the hypothesis of importation by infected animals or humans entering Sudan from the Congo Basin, and person-to-person transmission of virus, rather than transmission of indigenous virus from infected animals to humans. |
Humoral immunity to smallpox vaccines and monkeypox virus challenge; proteomic assessment and clinical correlations
Townsend MB , Keckler MS , Patel N , Davies DH , Felgner P , Damon IK , Karem KL . J Virol 2012 87 (2) 900-11 Despite the eradication of smallpox, Orthopoxviruses (OPV) remain public health concerns. Efforts to develop new therapeutics and vaccines for smallpox continue through their evaluation in animal models despite limited understanding of the specific correlates of protective immunity. Recent monkeypox virus challenge studies have established the Black-Tailed Prairie Dog (Cynomys ludovicianus) as a model of human systemic OPV infections. In this study, we assess the induction of humoral immunity in humans and prairie dogs receiving Dryvax(R), ACAM2000(R), or IMVAMUNE(R) vaccines and characterize the proteomic profile of immune recognition using ELISA, neutralization assays and protein microarrays. We confirm anticipated similarities of antigenic protein targets of smallpox vaccine-induced responses in humans and prairie dogs and identify several differences. Subsequent monkeypox intranasal infection of vaccinated prairie dogs results in a significant boost in humoral immunity characterized by a shift in reactivity of increased intensity to a broader range of OPV proteins. This work provides evidence of similarities between the vaccine response in prairie dogs and humans, which enhance the value of the prairie dog model system as an OPV vaccination model and offers novel findings that form a framework for examining humoral immune response induced by systemic infection of orthopoxviruses. |
Evaluation of the Tetracore Orthopox BioThreat(R) antigen detection assay using laboratory grown orthopoxviruses and rash illness clinical specimens
Townsend MB , Macneil A , Reynolds MG , Hughes CM , Olson VA , Damon IK , Karem KL . J Virol Methods 2012 187 (1) 37-42 The commercially available Orthopox BioThreat((R)) Alert assay for orthopoxvirus (OPV) detection is piloted. This antibody-based lateral-flow assay labels and captures OPV viral agents to detect their presence. Serial dilutions of cultured Vaccinia virus (VACV) and Monkeypox virus (MPXV) were used to evaluate the sensitivity of the Tetracore assay by visual and quantitative determinations; specificity was assessed using a small but diverse set of diagnostically relevant blinded samples from viral lesions submitted for routine OPV diagnostic testing. The BioThreat((R)) Alert assay reproducibly detected samples at concentrations of 10(7)pfu/ml for VACV and MPXV and positively identified samples containing 10(6)pfu/ml in 4 of 7 independent experiments. The assay correctly identified 9 of 11 OPV clinical samples and had only one false positive when testing 11 non-OPV samples. Results suggest applicability for use of the BioThreat((R)) Alert assay as a rapid screening assay and point of care diagnosis for suspect human monkeypox cases. |
The pox in the North American backyard: Volepox virus pathogenesis in California mice (Peromyscus californicus)
Gallardo-Romero NF , Drew CP , Weiss SL , Metcalfe MG , Nakazawa YJ , Smith SK , Emerson GL , Hutson CL , Salzer JS , Bartlett JH , Olson VA , Clemmons CJ , Davidson WB , Zaki SR , Karem KL , Damon IK , Carroll DS . PLoS One 2012 7 (8) e43881 Volepox virus (VPXV) was first isolated in 1985 from a hind foot scab of an otherwise healthy California vole (Microtus californicus). Subsequent surveys in San Mateo County, CA, revealed serological evidence suggesting that VPXV is endemic to this area, and a second viral isolate from a Pinyon mouse (Peromyscus truei) was collected in 1988. Since then, few studies have been conducted regarding the ecology, pathology, and pathogenicity of VPXV, and its prevalence and role as a potential zoonotic agent remain unknown. To increase our understanding of VPXV disease progression, we challenged 24 California mice (Peromyscus californicus) intranasally with 1.6x10(3) PFU of purified VPXV. By day five post infection (pi) we observed decreased activity level, conjunctivitis, ruffled hair, skin lesions, facial edema, and crusty noses. A mortality rate of 54% was noted by day eight pi. In addition, internal organ necrosis and hemorrhages were observed during necropsy of deceased or euthanized animals. Viral loads in tissues (brain, gonad, kidney, liver, lung, spleen, submandibular lymph node, and adrenal gland), bodily secretions (saliva, and tears), and excretions (urine, and/or feces) were evaluated and compared using real time-PCR and tissue culture. Viral loads measured as high as 2x10(9) PFU/mL in some organs. Our results suggest that VPXV can cause extreme morbidity and mortality within rodent populations sympatric with the known VPXV reservoirs. |
Progressive vaccinia: case description and laboratory-guided therapy with vaccinia immune globulin, ST-246 and CMX001
Lederman ER , Davidson W , Groff HL , Smith SK , Warkentien T , Li Y , Wilkins KA , Karem KL , Akondy RS , Ahmed R , Frace M , Shieh WJ , Zaki S , Hruby DE , Painter WP , Bergman KL , Cohen JI , Damon IK . J Infect Dis 2012 206 (9) 1372-85 Progressive vaccinia (PV) is a rare but potentially lethal complication which develops in smallpox vaccine recipients with severely impaired cellular immunity. We report a patient with PV requiring treatment with vaccinia immune globulin and who received two investigational agents, ST-246 and CMX001. We describe the various molecular, pharmacokinetic, and immunologic studies which provided guidance to escalate and then successfully discontinue therapy. Despite development of resistance to ST-246 during treatment, the patient had resolution of his PV. This case demonstrates the need for continued development of novel anti-orthopoxvirus pharmaceuticals and the importance of both intensive and timely clinical and laboratory support in management of PV. |
Factors affecting the likelihood of monkeypox's emergence and spread in the post-smallpox era
Reynolds MG , Carroll DS , Karem KL . Curr Opin Virol 2012 2 (3) 335-43 In 1980, the World Health Assembly announced that smallpox had been successfully eradicated as a disease of humans. The disease clinically and immunologically most similar to smallpox is monkeypox, a zoonosis endemic to moist forested regions in West and Central Africa. Smallpox vaccine provided protection against both infections. Monkeypox virus is a less efficient human pathogen than the agent of smallpox, but absent smallpox and the population-wide immunity engendered during eradication efforts, could monkeypox now gain a foothold in human communities? We discuss possible ecologic and epidemiologic limitations that could impede monkeypox's emergence as a significant pathogen of humans, and evaluate whether genetic constrains are sufficient to diminish monkeypox virus' capacity for enhanced specificity as a parasite of humans. |
Analysis of variola and vaccinia neutralization assays for smallpox vaccines
Hughes CM , Newman FK , Davidson WB , Olson VA , Smith SK , Holman RC , Yan L , Frey SE , Belshe RB , Karem KL , Damon IK . Clin Vaccine Immunol 2012 19 (7) 1116-8 Possible smallpox re-emergence drives research for third-generation vaccines that effectively neutralize variola virus. Comparison of neutralization assays using different substrates, variola and vaccinia (Dryvax and MVA), showed significantly different 90% neutralization titers; Dryvax underestimated while MVA overestimated variola neutralization. Third-generation vaccines may rely upon neutralization as a correlate of protection. |
Detection of North American orthopoxviruses by real time-PCR.
Gallardo-Romero NF , Velasco-Villa A , Weiss SL , Emerson GL , Carroll DS , Hughes CM , Li Y , Karem KL , Damon IK , Olson VA . Virol J 2011 8 313 The prevalence of North American orthopoxviruses in nature is unknown and may be more difficult to ascertain due to wide spread use of vaccinia virus recombinant vaccines in the wild. A real time PCR assay was developed to allow for highly sensitive and specific detection of North American orthopoxvirus DNA in animal tissues and bodily fluids. This method is based on the amplification of a 156 bp sequence within a myristylated protein, highly conserved within the North American orthopoxviruses but distinct from orthologous genes present in other orthopoxviruses. The analytical sensitivity was 1.1 fg for Volepox virus DNA, 1.99 fg for Skunkpox virus DNA, and 6.4 fg for Raccoonpox virus DNA with a 95% confidence interval. Our assay did not cross-react with other orthopoxviruses or ten diverse representatives of the Chordopoxvirinae subfamily. This new assay showed more sensitivity than tissue culture tests, and was capable of differentiating North American orthopoxviruses from other members of Orthopoxvirus. Thus, our assay is a promising tool for highly sensitive and specific detection of North American orthopoxviruses in the United States and abroad. |
Protection from smallpox: beyond immune biomarkers
Karem KL , Reynolds MG . Future Virol 2011 6 (6) 709-719 Since the eradication of smallpox, immunological studies of smallpox vaccine (vaccinia virus) have provided great gains in understanding immunology as well as depecting smallpox vaccine-derived immune responses. Despite this wealth of knowledge, two confounding problems remain. First, that surviving smallpox provides an individual with lifelong protective immunity, whereas vaccination may not, and second, that specific molecular correlates of protection against smallpox remain vague. Historical literature on smallpox and contemporary studies of other human infections with orthopoxviruses, such as monkeypox, indicate that vaccination may lower disease risks, but it does not provide complete protection against infection in all individuals. Factors impacting protective immunity include longevity of immunologic memory post-vaccination, challenge dose and differences in the challenge viruses, as well as route of exposure. This article discusses historical information regarding smallpox attack rates during outbreaks and contemporary views on aspects of vaccines and naturally occurring orthopoxvirus outbreaks as related to vaccine-derived immunity. 2011 Future Medicine Ltd. |
Establishment of the black-tailed prairie dog (Cynomys ludovicianus) as a novel animal model for comparing smallpox vaccines administered pre-exposure in both high and low-dose monkeypox challenges
Keckler MS , Carroll DS , Gallardo-Romero NF , Lash RR , Salzer JS , Weiss SL , Patel N , Clemmons CJ , Smith SK , Hutson CL , Karem KL , Damon IK . J Virol 2011 85 (15) 7683-98 The 2003 Monkeypox virus (MPXV) outbreak and subsequent laboratory studies demonstrated that the black-tailed prairie dog was susceptible to MPXV infection and that the ensuing rash illness was similar to human systemic orthopoxvirus (OPXV) infections - including a 7 to 9-day incubation period and likely, in some cases, a respiratory route of infection - which distinguishes this model from others. The need for safe and efficacious vaccines for both endemic and epidemic OPVX disease is important to protect an increasingly OPXV naive population. In this study, we tested current and investigational smallpox vaccines for safety, induction of anti-OPXV antibodies, and protection against mortality and morbidity in two MPXV challenges. None of the smallpox vaccines caused illness in this model and all vaccinated animals showed anti-OPXV antibody responses and neutralizing antibody. We tested vaccine efficacy by challenging the animals with 10(5) or 10(6) pfu Congo Basin MPXV 30 days post vaccination and evaluating morbidity and mortality. Our results demonstrate that vaccination with either Dryvax(R) or Acambis2000(R) protected the animals from death with no rash illness. Vaccination with IMVAMUNE(R) also protected the animals from death, albeit with (modified) rash illness. Based on the results of this study, we believe prairie dogs offer a novel and potentially useful small animal model for the safety and efficacy testing of smallpox vaccines in pre- and post-exposure vaccine testing, which is important for public health planning. |
Human monkeypox outbreak caused by novel virus belonging to Congo Basin clade, Sudan, 2005
Formenty P , Muntasir MO , Damon I , Chowdhary V , Opoka ML , Monimart C , Mutasim EM , Manuguerra JC , Davidson WB , Karem KL , Cabeza J , Wang S , Malik MR , Durand T , Khalid A , Rioton T , Kuong-Ruay A , Babiker AA , Karsani ME , Abdalla MS . Emerg Infect Dis 2010 16 (10) 1539-1545 To determine the outbreak source of monkeypox virus (MPXV) infections in Unity State, Sudan, in November 2005, we conducted a retrospective investigation. MPXV was identified in a sub-Sahelian savannah environment. Three case notification categories were used: suspected, probable, and confirmed. Molecular, virologic, and serologic assays were used to test blood specimens, vesicular swabs, and crust specimens obtained from symptomatic and recovering persons. Ten laboratory-confirmed cases and 9 probable cases of MPXV were reported during September-December 2005; no deaths occurred. Human-to-human transmission up to 5 generations was described. Our investigation could not fully determine the source of the outbreak. Preliminary data indicate that the MPXV strain isolated during this outbreak was a novel virus belonging to the Congo Basin clade. Our results indicate that MPXV should be considered endemic to the wetland areas of Unity State. This finding will enhance understanding of the ecologic niche for this virus. |
Physiologic reference ranges for captive black-tailed prairie dogs (Cynomys ludovicianus)
Keckler MS , Gallardo-Romero NF , Langham GL , Damon IK , Karem KL , Carroll DS . J Am Assoc Lab Anim Sci 2010 49 (3) 274-281 The black-tailed prairie dog (Cynomys ludovicianus) is a member of the order Rodentia and the family Sciuridae. Ecologically, prairie dogs are a keystone species in prairie ecology. This species is used as an animal model for human gallbladder disease and diseases caused by infection with Clostridium difficile, Yersinia pestis, Francisella tularensis, and most recently, Orthopoxvirus. Despite increasing numbers of prairie dogs used in research and kept as pets, few data are available on their baseline physiology in animal facility housing conditions. To establish baseline physiologic reference ranges, we designed a study using 18 wild-caught black-tailed prairie dogs. Telemetry data were analyzed to establish circadian rhythms for activity and temperature. In addition, hematologic and serum chemistry analyses were performed. Baseline measurements were used to establish the mean for each animal, which then were compiled and analyzed to determine the reference ranges. Here we present physiologic data on serum chemistry and hematology profiles, as well as weight, core body temperature, and daily activity patterns for black-tailed prairie dogs. These results reflect the use of multiple measurements from species- and age-matched prairie dogs and likely will be useful to ecologists, scientists interested in using this animal model in research, and veterinarians caring for pet prairie dogs. |
Dosage comparison of Congo Basin and West African strains of monkeypox virus using a prairie dog animal model of systemic orthopoxvirus disease
Hutson CL , Carroll DS , Self J , Weiss S , Hughes CM , Braden Z , Olson VA , Smith SK , Karem KL , Regnery RL , Damon IK . Virology 2010 402 (1) 72-82 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
Hutson CL , Abel JA , Carroll DS , Olson VA , Braden ZH , Hughes CM , Dillon M , Hopkins C , Karem KL , Damon IK , Osorio JE . PLoS One 2010 5 (1) e8912 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. |
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