Neville K. Kisalu

Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo

Studies on the burden of human monkeypox in the Democratic Republic of the Congo (DRC) were last conducted from 1981 to 1986. Since then, the population that is immunologically naïve to orthopoxviruses has increased significantly due to cessation of mass smallpox vaccination campaigns. To assess the current risk of infection, we analyzed human monkeypox incidence trends in a monkeypox-enzootic region. Active, population-based surveillance was conducted in nine health zones in central DRC. Epidemiologic data and biological samples were obtained from suspected cases. Cumulative incidence (per 10,000 population) and major determinants of infection were compared with data from active surveillance in similar regions from 1981 to 1986. Between November 2005 and November 2007, 760 laboratory-confirmed human monkeypox cases were identified in participating health zones. The average annual cumulative incidence across zones was 5.53 per 10,000 (2.18–14.42). Factors associated with increased risk of infection included: living in forested areas, male gender, age < 15, and no prior smallpox vaccination. Vaccinated persons had a 5.2-fold lower risk of monkeypox than unvaccinated persons (0.78 vs. 4.05 per 10,000). Comparison of active surveillance data in the same health zone from the 1980s (0.72 per 10,000) and 2006–07 (14.42 per 10,000) suggests a 20-fold increase in human monkeypox incidence. Thirty years after mass smallpox vaccination campaigns ceased, human monkeypox incidence has dramatically increased in rural DRC. Improved surveillance and epidemiological analysis is needed to better assess the public health burden and develop strategies for reducing the risk of wider spread of infection.

Endemic Human Monkeypox, Democratic Republic of Congo, 2001–2004

By analyzing vesicle fluids and crusted scabs from 136 persons with suspected monkeypox, we identified 51 cases of monkeypox by PCR, sequenced the hemagglutinin gene, and confirmed 94% of cases by virus culture. PCR demonstrated chickenpox in 61 patients. Coinfection with both viruses was found in 1 additional patient.

Propulsion of African trypanosomes is driven by bihelical waves with alternating chirality separated by kinks.

Trypanosoma brucei, a parasitic protist with a single flagellum, is the causative agent of African sleeping sickness. Propulsion of T. brucei was long believed to be by a drill-like, helical motion. Using millisecond differential interference-contrast microscopy and analyzing image sequences of cultured procyclic-form and bloodstream-form parasites, as well as bloodstream-form cells in infected mouse blood, we find that, instead, motility of T. brucei is by the propagation of kinks, separating left-handed and right-handed helical waves. Kink-driven motility, previously encountered in prokaryotes, permits T. brucei a helical propagation mechanism while avoiding the large viscous drag associated with a net rotation of the broad end of its tapering body. Our study demonstrates that millisecond differential interference-contrast microscopy can be a useful tool for uncovering important short-time features of microorganism locomotion.

Structure-Function Analysis of Dynein Light Chain 1 Identifies Viable Motility Mutants in Bloodstream-Form Trypanosoma brucei

ABSTRACT The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei. Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness.

Genomic Variability of Monkeypox Virus among Humans, Democratic Republic of the Congo

Health authorities should be vigilant for this rapidly evolving virus.

Mouse infection and pathogenesis by Trypanosoma brucei motility mutants

The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. In the mammalian host, parasite motility is suspected to contribute to infection and disease pathogenesis. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. We recently identified a bloodstream‐form motility mutant in 427‐derived T. brucei in which point mutations in the LC1 dynein subunit disrupt propulsive motility but do not affect viability. These mutants have an actively beating flagellum, but cannot translocate. Here we demonstrate that the LC1 point mutant fails to show enhanced cell motility upon increasing viscosity of the surrounding medium, which is a hallmark of wild type T. brucei, thus indicating that motility of the mutant is fundamentally altered compared with wild type cells. We next used the LC1 point mutant to assess the influence of trypanosome motility on infection in mice. Wesurprisingly found that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. Infection time‐course, maximum parasitaemia, number of waves of parasitaemia, clinical features and disease outcome are indistinguishable between motility mutant and control parasites. Our studies provide an important step toward understanding the contribution of parasite motility to infection and a foundation for future investigations of T. brucei interaction with the mammalian host.

Pathogen-Host Associations and Predicted Range Shifts of Human Monkeypox in Response to Climate Change in Central Africa

Climate change is predicted to result in changes in the geographic ranges and local prevalence of infectious diseases, either through direct effects on the pathogen, or indirectly through range shifts in vector and reservoir species. To better understand the occurrence of monkeypox virus (MPXV), an emerging Orthopoxvirus in humans, under contemporary and future climate conditions, we used ecological niche modeling techniques in conjunction with climate and remote-sensing variables. We first created spatially explicit probability distributions of its candidate reservoir species in Africas Congo Basin. Reservoir species distributions were subsequently used to model current and projected future distributions of human monkeypox (MPX). Results indicate that forest clearing and climate are significant driving factors of the transmission of MPX from wildlife to humans under current climate conditions. Models under contemporary climate conditions performed well, as indicated by high values for the area under the receiver operator curve (AUC), and tests on spatially randomly and non-randomly omitted test data. Future projections were made on IPCC 4th Assessment climate change scenarios for 2050 and 2080, ranging from more conservative to more aggressive, and representing the potential variation within which range shifts can be expected to occur. Future projections showed range shifts into regions where MPX has not been recorded previously. Increased suitability for MPX was predicted in eastern Democratic Republic of Congo. Models developed here are useful for identifying areas where environmental conditions may become more suitable for human MPX; targeting candidate reservoir species for future screening efforts; and prioritizing regions for future MPX surveillance efforts.

Cytokine modulation correlates with severity of monkeypox disease in humans

BACKGROUND Human monkeypox is a zoonotic disease endemic to parts of Africa. Similar to other orthopoxviruses, virus and host have considerable interactions through immunomodulation. These interactions likely drive the establishment of a productive infection and disease progression, resulting in the range of disease presentations and case fatality rates observed for members of the Orthopoxvirus genus. OBJECTIVES Much of our understanding about the immune response to orthopoxvirus infection comes from either in vitro or in vivo studies performed in small animals or non-human primates. Here, we conducted a detailed assessment of cytokine responses to monkeypox virus using serum from acutely ill humans collected during monkeypox active disease surveillance (2005-2007) in the Democratic Republic of the Congo. STUDY DESIGN Nineteen serum samples that were from patients with confirmed monkeypox virus infections were selected for cytokine profiling. Cytokine profiling was performed on the Bio-Rad Bioplex 100 system using a 30-plex human cytokine panel. RESULTS Cytokine profiling revealed elevated cytokine concentrations in all samples. Overproduction of certain cytokines (interleukin [IL]-2R, IL-10, and granulocyte macrophage-colony stimulating factor were observed in patients with serious disease (defined as >250 lesions based on the World Health Organization scoring system). CONCLUSIONS The data suggest that cytokine modulation affects monkeypox disease severity in humans.

Parasite motility is critical for virulence of African trypanosomes

African trypanosomes, Trypanosoma brucei spp., are lethal pathogens that cause substantial human suffering and limit economic development in some of the world’s most impoverished regions. The name Trypanosoma (“auger cell”) derives from the parasite’s distinctive motility, which is driven by a single flagellum. However, despite decades of study, a requirement for trypanosome motility in mammalian host infection has not been established. LC1 is a conserved dynein subunit required for flagellar motility. Prior studies with a conditional RNAi-based LC1 mutant, RNAi-K/R, revealed that parasites with defective motility could infect mice. However, RNAi-K/R retained residual expression of wild-type LC1 and residual motility, thus precluding definitive interpretation. To overcome these limitations, here we generate constitutive mutants in which both LC1 alleles are replaced with mutant versions. These double knock-in mutants show reduced motility compared to RNAi-K/R and are viable in culture, but are unable to maintain bloodstream infection in mice. The virulence defect is independent of infection route but dependent on an intact host immune system. By comparing different mutants, we also reveal a critical dependence on the LC1 N-terminus for motility and virulence. Our findings demonstrate that trypanosome motility is critical for establishment and maintenance of bloodstream infection, implicating dynein-dependent flagellar motility as a potential drug target.