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.

One of the most commonly used molecular test for malaria diagnosis is the polymerase chain reaction (PCR)-based amplification of the 18S ribosomal DNA (rDNA) gene. Published diagnostic assays based on the 18S gene include the "gold standard" nested assay, semi-nested multiplex assay, and one tube multiplex assay. To our knowledge, no one has reported whether the two multiplex methods are better at detecting mixed Plasmodium infections compared to the nested assay using known quantities of DNA in experimentally mixed cocktails.

Adverse events upon smallpox vaccination with fully-replicative strains of Vaccinia virus (VACV) comprise an array of clinical manifestations that occur primarily in immunocompromised patients leading to significant host morbidity/mortality. The expansion of immune-suppressed populations and the possible release of Variola virus as a bioterrorist act have given rise to concerns over vaccination complications should more widespread vaccination be reinitiated. Our goal was to evaluate the components of the host immune system that are sufficient to prevent morbidity/mortality in a murine model of tail scarification, which mimics immunological and clinical features of smallpox vaccination in humans. Infection of C57BL/6 wild-type mice led to a strictly localized infection, with complete viral clearance by day 28 p.i. On the other hand, infection of T and B-cell deficient mice (Rag1(-/-)) produced a severe disease, with uncontrolled viral replication at the inoculation site and dissemination to internal organs. Infection of B-cell deficient animals (µMT) produced no mortality. However, viral clearance in µMT animals was delayed compared to WT animals, with detectable viral titers in tail and internal organs late in infection. Treatment of Rag1(-/-) with rabbit hyperimmune anti-vaccinia serum had a subtle effect on the morbidity/mortality of this strain, but it was effective in reduce viral titers in ovaries. Finally, NUDE athymic mice showed a similar outcome of infection as Rag1(-/-), and passive transfer of WT T cells to Rag1(-/-) animals proved fully effective in preventing morbidity/mortality. These results strongly suggest that both T and B cells are important in the immune response to primary VACV infection in mice, and that T-cells are required to control the infection at the inoculation site and providing help for B-cells to produce antibodies, which help to prevent viral dissemination. These insights might prove helpful to better identify individuals with higher risk of complications after infection with poxvirus.

Molluscum contagiosum virus (MCV) causes an innocuous yet persistent skin infection in immunocompetent individuals and is spread by contact with lesions. Studies point to atopic dermatitis (AD) as a risk factor for MCV infection; however, there are no longitudinal studies that have evaluated this hypothesis.

Previous work suggests that Brazilian Plasmodium falciparum has limited genetic diversity and a history of bottlenecks, multiple reintroductions due to human migration, and clonal expansions. We hypothesized that Brazilian P. falciparum would exhibit clonal structure. We examined isolates collected across two decades from Amapá, Rondônia, and Pará state (n = 190). By examining more microsatellites markers on more chromosomes than previous studies, we hoped to define the extent of low diversity, linkage disequilibrium, bottlenecks, population structure, and parasite migration within Brazil. We used retrospective genotyping of samples from the 1980s and 1990s to explore the population genetics of SP resistant dhfr and dhps alleles. We tested an existing hypothesis that the triple mutant dhfr mutations 50R/51I/108N and 51I/108N/164L developed in southern Amazon from a single origin of common or similar parasites. We found that Brazilian P. falciparum had limited genetic diversity and isolation by distance was rejected, which suggests it underwent bottlenecks followed by migration between sites. Unlike Peru, there appeared to be gene flow across the Brazilian Amazon basin. We were unable to divide parasite populations by clonal lineages and pairwise FST were common. Most parasite diversity was found within sites in the Brazilian Amazon, according to AMOVA. Our results challenge the hypothesis that triple mutant alleles arose from a single lineage in the Southern Amazon. SP resistance, at both the double and triple mutant stages, developed twice and potentially in different regions of the Brazilian Amazon. We would have required samples from before the 1980s to describe how SP resistance spread across the basin or describe the complex internal migration of Brazilian parasites after the colonization efforts of past decades. The Brazilian Amazon basin may have sufficient internal migration for drug resistance reported in any particular region to rapidly spread to other parts of basin under similar drug pressure.

In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL). The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR) assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) peruviana and L. (V.) lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST). In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST) data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America.

In areas where health resources are limited, community participation in the recognition and reporting of disease hazards is critical for the identification of outbreaks. This is particularly true for zoonotic diseases such as monkeypox that principally affect people living in remote areas with few health services. Here we report the findings of an evaluation measuring the effectiveness of a film-based community outreach program designed to improve the understanding of monkeypox symptoms, transmission and prevention, by residents of the Republic of the Congo (ROC) who are at risk for disease acquisition.

Cowpox virus (CPXV) is described as the source of the first vaccine used to prevent the onset and spread of an infectious disease. It is one of the earliest described members of the genus Orthopoxvirus, which includes the viruses that cause smallpox and monkeypox in humans. Both the historic and current literature describe "cowpox" as a disease with a single etiologic agent. Genotypic data presented herein indicate that CPXV is not a single species, but a composite of several (up to 5) species that can infect cows, humans, and other animals. The practice of naming agents after the host in which the resultant disease manifests obfuscates the true taxonomic relationships of "cowpox" isolates. These data support the elevation of as many as four new species within the traditional "cowpox" group and suggest that both wild and modern vaccine strains of Vaccinia virus are most closely related to CPXV of continental Europe rather than the United Kingdom, the homeland of the vaccine.

Members of the genus Yatapoxvirus, which include Tanapox virus (TPV) and Yaba monkey tumor virus, infect primates including humans. Two strains of TPV isolated 50 years apart from patients infected from the equatorial region of Africa have been sequenced. The original isolate from a human case in the Tana River Valley, Kenya, in 1957 (TPV-Kenya) and an isolate from an infected traveler in the Republic of Congo in 2004 (TPV-RoC). Although isolated 50 years apart the genomes were highly conserved. The genomes differed at only 35 of 144,565 nucleotide positions (99.98% identical). We predict that TPV-RoC encodes 155 ORFs, however a single transversion (at nucleotide 10241) in TPV-Kenya resulted in the coding capacity for two predicted ORFs (11.1L and 11.2L) in comparison to a single ORF (11L) in TPV-RoC. The genomes of TPV are A+T rich (73%) and 96% of the sequence encodes predicted ORFs. Comparative genomic analysis identified several features shared with other chordopoxviruses. A conserved sequence within the terminal inverted repeat region that is also present in the other members of the Yatapoxviruses as well as members of the Capripoxviruses, Swinepox virus and an unclassified Deerpox virus suggests the existence of a conserved near-terminal sequence secondary structure. Two previously unidentified gene families were annotated that are represented by ORF TPV28L, which matched homologues in certain other chordopoxviruses, and TPV42.5L, which is highly conserved among currently reported chordopoxvirus sequences.

Although the World Health Organization (WHO) declared global smallpox eradicated in 1980, concerns over emergent poxvirus infections have increased. Most poxvirus infections are zoonotic; exploring their genetic diversity will illuminate the genetic and evolutionary aspects of poxvirus infections, ecology, and epidemiology. In recent decades, several strains of the orthopoxvirus vaccinia virus (VACV) have been isolated throughout Brazil, including genetically distinct isolates within the same outbreak. To further investigate the diversity and origins of these viruses, we analyzed molecular data from 8 Brazilian VACV isolates and compared several genes involved in virus structure and pathogenicity. Genetic variation among isolates suggests that ancestral Brazilian VACVs existed before the beginning of the WHO smallpox eradication vaccination campaigns and that these viruses continue to circulate.

Several of the intended Plasmodium falciparum vaccine candidate antigens are highly polymorphic and could render a vaccine ineffective if their antigenic sites were not represented in the vaccine. In this study, characterization of genetic variability was performed in major B and T-cell epitopes within vaccine candidate antigens in isolates of P. falciparum from Peru.

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 104 pfu (2× LD50) or 106 pfu (170× LD50) 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× LD50, but not the 170× LD5 challenge. In the 2× LD50 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.

Several tyrosine kinase inhibitors (TKIs) developed as anti-cancer drugs, also have anti-viral activity due to their ability to disrupt productive replication and dissemination in infected cells. Consequently, such drugs are attractive candidates for "repurposing" as anti-viral agents. However, clinical evaluation of therapeutics against infectious agents associated with high mortality, but low or infrequent incidence, is often unfeasible. The United States Food and Drug Administration formulated the "Animal Rule" to facilitate use of validated animal models for conducting anti-viral efficacy studies.

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.

Since the eradication of smallpox, there have been increases in poxvirus infections and the emergence of several novel poxviruses that can infect humans and domestic animals. In 2015, a novel poxvirus was isolated from a resident of Alaska. Diagnostic testing and limited sequence analysis suggested this isolate was a member of the Orthopoxvirus (OPXV) genus but was highly diverged from currently known species, including Akhmeta virus. Here, we present the complete 210,797 bp genome sequence of the Alaska poxvirus isolate, containing 206 predicted open reading frames. Phylogenetic analysis of the conserved central region of the genome suggested the Alaska isolate shares a common ancestor with Old World OPXVs and is diverged from New World OPXVs. We propose this isolate as a member of a new OPXV species, Alaskapox virus (AKPV). The AKPV genome contained host range and virulence genes typical of OPXVs but lacked homologs of C4L and B7R, and the hemagglutinin gene contained a unique 120 amino acid insertion. Seven predicted AKPV proteins were most similar to proteins in non-OPXV Murmansk or NY_014 poxviruses. Genomic analysis revealed evidence suggestive of recombination with Ectromelia virus in two putative regions that contain seven predicted coding sequences, including the A-type inclusion protein.

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.

In the spring of 2006, four human cases of parapoxvirus infections in Missouri residents were reported to the Centers for Disease Control and Prevention (CDC), two of which were initially diagnosed as cutaneous anthrax. This investigation was conducted to determine the level of recognition of zoonotic parapoxvirus infections and prevention measures, the degree to which veterinarians may be consulted on human infections and what forces were behind this perceived increase in reported infections. Interviews were conducted and clinical and environmental sampling was performed. Swab and scab specimens were analyzed by real-time polymerase chain reaction (PCR), whereas serum specimens were evaluated for parapoxvirus antibodies. Three case patients were found to have fed ill juvenile animals without using gloves. Forty-six percent of veterinarians reported having been consulted regarding suspected human orf infections. Orf virus DNA was detected from five of 25 asymptomatic sheep. Analysis of extracellular envelope gene sequences indicated that sheep and goat isolates clustered in a species-preferential fashion. Parapoxvirus infections are common in Missouri ruminants and their handlers. Infected persons often do not seek medical care; some may seek advice from veterinarians rather than physicians. The initial perception of increased incidence in Missouri may have arisen from a reporting artifact stemming from heightened concern about anthrax. Asymptomatic parapoxvirus infections in livestock may be common and further investigation warranted.

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.

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.

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.