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Chikungunya, a mosquito-borne viral disease caused by the chikungunya virus (CHIKV), causes a significant global health burden, and there is currently no approved vaccine to prevent chikungunya disease. In this study, the safety and immunogenicity of a CHIKV mRNA vaccine candidate (mRNA-1388) were evaluated in healthy participants in a CHIKV-nonendemic region. This phase 1, first-in-human, randomized, placebo-controlled, dose-ranging study enrolled healthy adults (ages 18–49 years) between July 2017 and March 2019 in the United States. Participants were randomly assigned (3:1) to receive 2 intramuscular injections 28 days apart with mRNA-1388 in 3 dose-level groups (25 μg, 50 μg, and 100 μg) or placebo and were followed for up to 1 year. Safety (unsolicited adverse events [AEs]), tolerability (local and systemic reactogenicity; solicited AEs), and immunogenicity (geometric mean titers [GMTs] of CHIKV neutralizing and binding antibodies) of mRNA-1388 versus placebo were evaluated. Sixty participants were randomized and received ≥ 1 vaccination; 54 (90 %) completed the study. mRNA-1388 demonstrated favorable safety and reactogenicity profiles at all dose levels. Immunization with mRNA-1388 induced substantial and persistent humoral responses. Dose-dependent increases in neutralizing antibody titers were observed; GMTs (95 % confidence intervals [CIs]) at 28 days after dose 2 were 6.2 (5.1–7.6) for mRNA-1388 25 μg, 53.8 (26.8–108.1) for mRNA-1388 50 μg, 92.8 (43.6–197.6) for mRNA-1388 100 μg, and 5.0 (not estimable) for placebo. Persistent humoral responses were observed up to 1 year after vaccination and remained higher than placebo in the 2 higher mRNA-1388 dose groups. The development of CHIKV-binding antibodies followed a similar trend as that observed with neutralizing antibodies. mRNA-1388, the first mRNA vaccine against CHIKV, was well tolerated and elicited substantial and long-lasting neutralizing antibody responses in healthy adult participants in a nonendemic region. ClinicalTrials.gov: NCT03325075.

OBJECTIVES/GOALS: Whole-genome viral sequencing is vital to inform public health and study evolution. Arboviruses evolve in vectors, reservoir hosts, and humans, and require surveillance at all points. We developed a new rigorous method of sequencing that captures whole viral genomes in field-collected and clinical samples. METHODS/STUDY POPULATION: ClickSeq is a novel method of Next Generation Sequencing (NGS) library synthesis using azido-nucleotides to terminate reverse transcription. The cDNA generated can be ligated to sequencing and indexing primers at room temperature using copper (Cu I) and vitamin C. With this approach, we designed primers located ~250 bp apart along the genomes of the arboviruses Chikungunya 37797, Zika Dakar, Yellow Fever Asibi, Dengue serotype 2, West Nile 385-99, and St. Louis Encephalitis Virus (SLEV) clade II. We tested this method with varying viral titers: lab-infected mosquito pools, field-collected mosquito pools from a Texas West Nile and SLEV outbreak, and patient isolates from a Pakistani CHIKV outbreak. The cDNA was sequenced in the UTMB NGS Core and aligned using bowtie. RESULTS/ANTICIPATED RESULTS: The use of a single protocol to capture whole viral genomes including UTRs for multiple viruses from different sample collection styles is ideal for arboviruses. Primers for multiple viruses were pooled and used to sequence mosquito pools. The Tiled ClickSeq method captured whole viral genomes without the need for host depletion. UTRs were captured even when the viral strain used for primer design differed from the resulting strain. Discreet variants were captured in both the hypervariable nsP3 region and the UTR in the patient isolates from the CHIKV outbreak compared to the 2017 outbreak. Texas WNV and SLEV outbreaks are now defined from the 2020 outbreak and can be further tracked to update public health measures and understand viral evolution. DISCUSSION/SIGNIFICANCE: UTRs impact both human and mosquito fitness, leading to further outbreaks. Tiled ClickSeq aims to capture whole viral genomes with a method and cost that can be implemented by public health researchers to understand disease evolution as it happens to update both public health and basic virology to the effects of evolution on arboviruses.

Key Points Many recent viral pandemics have been attributed to the ability of some RNA viruses, for example HIV, dengue virus and possibly the severe acute respiratory syndrome (SARS) coronavirus, to change their host range to include humans. The authors discuss the mechanisms of host-range alteration used by a selection of viruses, including Venezuelan equine and Japanese encephalitis viruses (VEEV and JEV, respectively), dengue virus and West Nile virus (WNV). Venezuelan equine encephalitis (VEE) was first recognized as a disease of horses, donkeys and mules in northern South America during the mid 1930s, but there has been renewed interest in this virus because of its potential as a biological weapon. Molecular analysis of epidemic strains — which exploit horses for amplification — and comparison with strains that do not cause epidemic disease, have shown that a few amino-acid mutations can affect host-range alteration. Changes on the surface of the VEE virion seem to be important for these host range changes. JEV causes epidemics of encephalitis in India, Korea, China, South-East Asia and Indonesia. The disease affects children, and is associated with a mortality rate of greater than 20%. However, unlike VEEV, there is no evidence that JEV undergoes mutation and selection to replicate in different hosts. Pigs amplify transmission in peridomestic settings, and migratory birds have a role in dispersion of JEV. Although different genotypes have been isolated, their relevance to pathology and host range is unclear. WNV is now endemic in the United States after first emerging in New York in 1999. WNV has a very broad host range. Forty-nine species of mosquitoes and ticks, and 225 species of birds are susceptible to infection. Other hosts include horses, cattle, llamas, alligators, cats, dogs, wolves and sheep. Transmission of WNV among these species has not been reported. Although humans are probably dead-end hosts, infection with WNV can cause severe disease. Dengue viruses are very important human arboviral pathogens and use humans as reservoir hosts. Aedes aegypti and Aedes albopictus mosquitoes are the most common vectors in urban settings. It is thought that the human epidemic form of dengue virus evolved in the last 2000 years, and genetic analysis indicates that mutations have resulted in adaptation to the urban mosquito host. However, links between mutations and human pathogenicity have not been established. Finally, the authors discuss how host-range changes can be studied experimentally. Cell-culture model systems can be used to find mutations that correlate with virus fitness and adaptation in different host strains. Viruses that replicate in useful laboratory animal models can also be studied in whole animal hosts. Many pandemics have been attributed to the ability of some RNA viruses to change their host range to include humans. Here, we review the mechanisms of disease emergence that are related to the host-range specificity of selected mosquito-borne alphaviruses and flaviviruses. We discuss viruses of medical importance, including Venezuelan equine and Japanese encephalitis viruses, dengue viruses and West Nile viruses.

Cross-neutralization is generally a prerequisite for cross-protection of vaccines against diseases caused by heterologous viruses. Using sera obtained from a randomized clinical phase 3 trial in adults, we investigated the cross-neutralization activity of VLA1553, a vaccine recently approved to prevent chikungunya disease. Analysed in a plaque reduction neutralization test, the three major chikungunya virus (CHIKV) lineages, namely the East Central South African, the West African, and the Asian lineage, were inhibited by CHIKV-specific neutralizing antibodies present in the sera from vaccinated humans. This effect was independent of the time elapsed since vaccination. Moreover, the magnitude of the immune response was similar to the antibody levels detected in sera from convalescent chikungunya patients. Thus, VLA1553 has the potential to diminish the burden of chikungunya disease on a global scale.Trial registration: ClinicalTrials.gov identifier: NCT04546724.

Venezuelan equine encephalitis virus (VEEV) remains a naturally emerging disease threat as well as a highly developed biological weapon. Recently, progress has been made in understanding the complex ecological and viral genetic mechanisms that coincide in time and space to generate outbreaks. Enzootic, equine avirulent, serotype ID VEEV strains appear to alter their serotype to IAB or IC, and their vertebrate and mosquito host range, to mediate repeated VEE emergence via mutations in the E2 envelope glycoprotein that represent convergent evolution. Adaptation to equines results in highly efficient amplification, which results in human disease. Although epizootic VEEV strains are opportunistic in their use of mosquito vectors, the most widespread outbreaks appear to involve specific adaptation to Ochlerotatus taeniorhynchus , the most common vector in many coastal areas. In contrast, enzootic VEEV strains are highly specialized and appear to utilize vectors exclusively in the Spissipes section of the Culex ( Melanoconion ) subgenus.

In Senegal, chikungunya virus (CHIKV) is maintained in a sylvatic cycle and causes sporadic cases or small outbreaks in rural areas. However, little is known about the influence of the environment on its transmission. To address the question, 120 villages were randomly selected in the Kedougou region of southeastern Senegal. In each selected village, 10 persons by randomly selected household were sampled and tested for specific anti-CHIKV IgG antibodies by ELISA. We investigated the association of CHIKV seroprevalence with environmental variables using logistic regression analysis and the spatial correlation of village seroprevalence based on semivariogram analysis. Fifty-four percent (51%–57%) of individuals sampled during the survey tested positive for CHIKV-specific IgG. CHIKV seroprevalence was significantly higher in populations living close to forested areas (Normalized Difference Vegetation Index (NDVI), Odds Ratio (OR) = 1.90 (1.42–2.57)), and was negatively associated with population density (OR = 0.76 (0.69–0.84)). In contrast, in gold mining sites where population density was >400 people per km2, seroprevalence peaked significantly among adults (46% (27%–67%)) compared to all other individuals (20% (12%–31%)). However, traditional gold mining activities significantly modify the transmission dynamic of CHIKV, leading to a potential increase of the risk of human exposition in the region.

RNA viruses are notorious for their genetic plasticity and propensity to exploit new host-range opportunities, which can lead to the emergence of human disease epidemics such as severe acute respiratory syndrome, AIDS, dengue, and influenza. However, the mechanisms of host-range change involved in most of these viral emergences, particularly the genetic mechanisms of adaptation to new hosts, remain poorly understood. We studied the emergence of Venezuelan equine encephalitis virus (VEEV), an alphavirus pathogen of people and equines that has had severe health and economic effects in the Americas since the early 20th century. Between epidemics, VEE disappears for periods up to decades, and the viral source of outbreaks has remained enigmatic. Combined with phylogenetic analyses to predict mutations associated with a 1992-1993 epidemic, we used reverse genetic studies to identify an envelope glycoprotein gene mutation that mediated emergence. This mutation allowed an enzootic, equine-avirulent VEEV strain, which circulates among rodents in nearby forests to adapt for equine amplification. RNA viruses including alphaviruses exhibit high mutation frequencies. Therefore, ecological and epidemiological factors probably constrain the frequency of VEE epidemics more than the generation, via mutation, of amplification-competent (high equine viremia) virus strains. These results underscore the ability of RNA viruses to alter their host range, virulence, and epidemic potential via minor genetic changes. VEE also demonstrates the unpredictable risks to human health of anthropogenic changes such as the introduction of equines and humans into habitats that harbor zoonotic RNA viruses.

In 1993 and 1996, subtype IE Venezuelan equine encephalitis (VEE) virus caused epizootics in the Mexican states of Chiapas and Oaxaca. Previously, only subtype IAB and IC VEE virus strains had been associated with major outbreaks of equine and human disease. The IAB and IC epizootics are believed to emerge via adaptation of enzootic (sylvatic, equine-avirulent) strains for high titer equine viremia that results in efficient infection of mosquito vectors. However, experimental equine infections with subtype IE equine isolates from the Mexican outbreaks demonstrated neuro-virulence but little viremia, inconsistent with typical VEE emergence mechanisms. Therefore, we hypothesized that changes in the mosquito vector host range might have contributed to the Mexican emergence. To test this hypothesis, we evaluated the susceptibility of the most abundant mosquito in the deforested Pacific coastal locations of the VEE outbreaks and a proven epizootic vector, Ochlerotatus taeniorhynchus. The Mexican epizootic equine isolates exhibited significantly greater infectivity compared with closely related enzootic strains, supporting the hypothesis that adaptation to an efficient epizootic vector contributed to disease emergence. Reverse genetic studies implicated a Ser → Asn substitution in the E2 envelope glycoprotein as the major determinant of the increased vector infectivity phenotype. Our findings underscore the capacity of RNA viruses to alter their vector host range through minor genetic changes, resulting in the potential for disease emergence.

Nadia A Fernández-Santos,1,2,* Mario A Rodríguez-Pérez,1,* Sofía Segovia-Mancillas,1,3,* Luis L Rodríguez,4 Sarah A Hamer,5 Gabriel L Hamer,2 Fabián Correa-Morales,6 Susano Medina-Jaramillo,7 Maria Gabriela Palacios-Mendoza,8 Epigmenio Cruz-Aldán,9 Gabriela del Carmen Rodriguez-Dominguez,10 Carlos H Gomez-Hernandez,10 Arturo Larraga-Guillén,11 Irene López González,11 Luis M Rodríguez-Martínez,12 Aldo I Ortega-Morales,13 Ma Isabel Salazar,14 Héctor Enrique Valdez-Gómez,11 Miguel A Márquez Ruiz,15 Maria J Perteguer,16 Benjamín Gastón Gómez-Gordillo,17 Jesús A Aguilar-Durán,1 Ingeborg D Becker Fauser,18 Scott C Weaver,19 Michael J Turell,20 Laura D Kramer,21,* Jose Guillermo Estrada-Franco1,* 1Instituto Politécnico Nacional, Centro de Biotecnología Genómica, Reynosa, Tamaulipas, Mexico; 2Department of Entomology, Texas A&M University, College Station, TX, USA; 3Facultad de Medicina, Universidad México Americana del Norte, Reynosa, Tamaulipas, Mexico; 4Foreign Animal Disease Research Unit, USDA/ARS Plum Island Animal Disease Center, Greenport, NY, USA; 5Department of Veterinary Integrative Biosciences, College Station, TX, USA; 6Centro Nacional de Programas Preventivos y Control de Enfermedades, Secretaría de Salud, Ciudad de México, Mexico; 7Academia Veterinaria Mexicana, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Ciudad de México, Mexico; 8Zoologico Miguel Alvarez del Toro (ZOOMAT), Tuxtla Gutierrez, Chiapas, Mexico; 9Programas Ambientales Grupo Libera, Merida, Yucatán, Mexico; 10Laboratorio Estatal de Salud, Secretaria de Salud de Chiapas, Tuxtla Gutierrez, Chiapas, Mexico; 11CPA-SENASICA, Comisión México-Estados Unidos para la Prevención de la Fiebre Aftosa y otras Enfermedades Exóticas de los Animales, Ciudad de México, Mexico; 12Centro de Estudios e Investigaciones Interdisciplinarios, Universidad Autónoma de Coahuila, Saltillo, Coahuila, Mexico; 13Universidad Autónoma Agraria Antonio Narro Unidad Laguna, Torreón, Coahuila, Mexico; 14Laboratorio Nacional de Vacunología y Virus Tropicales, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Ciudad de México, Mexico; 15Departamento de Postgrado en Medicina Aviar, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Ciudad de México, Mexico; 16Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain; 17Comisión Nacional de Áreas Naturales Protegidas (CONANP), Palacio Federal 3er. Piso, Segunda Oriente-Norte, Tuxtla Gutiérrez, Chiapas, C.P. 29000, Mexico; 18Universidad Nacional Autónoma de México, Facultad de Medicina, Departamento de Medicina Experimental, Hospital General de México, Ciudad de México, Mexico; 19World Reference Center for Emerging Viruses and Arboviruses, Institute for Human Infections and Immunity, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA; 20VectorID LLC, Frederick, MD, USA; 21School of Public Health, University of the State of New York at Albany, Albany, NY, USA*These authors contributed equally to this workCorrespondence: Jose Guillermo Estrada-Franco, Instituto Politécnico Nacional, Centro de Biotecnología Genómica, Reynosa, Tamaulipas, Mexico, Tel +525534535428, Email jestradaf@ipn.mx Laura D Kramer, School of Public Health, University of the State of New York at Albany, Albany, NY, USA, Tel +15183222706, Email ldkculex@gmail.comAbstract: Zoonotic pathogens such as arboviruses, arenaviruses, filoviruses, coronaviruses, highly pathogenic Avian Influenza A (H5N1) viruses, vesiculoviruses, and many others are emerging and reemerging worldwide, jeopardizing global veterinary and public health. Parasitic diseases such as visceral and cutaneous leishmaniasis, trypanosomiasis (Trypanosoma cruzi), myiasis, and river blindness (Onchocerca volvulus) are also paramount for public health in the Americas and elsewhere. In the fall 2024, a group of experts convened in Chiapas, Mexico, for the Fourth Mesoamerican Symposium “Dr. Roberto Navarro López” on Arboviruses and Emerging Zoonotic Diseases. Here, we highlight the importance of some zoonotic pathogens and parasites affecting human health that are being impacted by anthropogenic activities. In this context, there are drivers such as changes in climate and landscape transformations, unsound agricultural practices, and wildlife niche replacement delivering numerous opportunities for zoonotic pathogens to emerge and threaten human health and food security.Keywords: zoonosis, arboviruses, emerging diseases, re-emerging diseases, symposium, training course

Eastern equine encephalitis virus (EEEV) produces the most severe human arboviral diseases in the United States, with mortality rates of 30%-70%. Vasculitis associated with microhemorrhages in the brain dominates the pathological picture in fatal human eastern equine encephalitis, and neuronal cell death is detectable during the late stage of the disease. We describe use of the golden hamster to study EEEV-induced acute vasculitis and encephalitis. In hamsters, EEEV replicates in visceral organs, produces viremia, and penetrates the brain. The pathological manifestations and antigen distribution in the brain of a hamster are similar to those described in human cases of EEEV.