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Salmonella myovirus SPN3US has a T = 27 capsid composed of >50 different gene products, including many that are packaged along with the 240 kb genome and ejected into the host cell. Recently, we showed that an essential phage-encoded prohead protease gp245 is responsible for cleavage of proteins during SPN3US head assembly. This proteolytic maturation step induces major changes in precursor head particles, enabling them to expand and undergo genome packaging. To comprehensively define the composition of the mature SPN3US head and elucidate how it is modified by proteolysis during assembly, we conducted tandem mass spectrometry analysis of purified virions and tailless heads. Fourteen protease cleavage sites were identified in nine proteins, including eight sites not previously identified in head proteins in vivo. Among these was the maturation cleavage site of gp245 which was identical to the autocleavage site we had previously identified in purified recombinant gp245. Our findings underscore the value of employing multiple mass spectrometry-based experimental strategies as a way to enhance the detection of head protein cleavage sites in tailed phages. In addition, our results have identified a conserved set of head proteins in related giant phages that are similarly cleaved by their respective prohead proteases, suggesting that these proteins have important roles in governing the formation and function of large icosahedral capsids.

Background The global impact of Zika virus in Latin America has drawn renewed attention to circulating mosquito-borne viruses in this region, such as dengue and chikungunya. Our objective was to assess socio-ecological factors associated with Aedes mosquito vector density as a measure of arbovirus transmission risk in three cities of potentially recent Zika virus introduction: Ibagué, Colombia; Manta, Ecuador; and Posadas, Argentina, in order to inform disease mitigation strategies. Methods We sampled Aedes mosquito populations in a total of 1086 households, using indoor and peridomestic mosquito collection methods, including light traps, resting traps, traps equipped with chemical attractant and aspirators. For each sampled household, we collected socio-economic data using structured questionnaires and data on microenvironmental conditions using iButton data loggers. Results A total of 3230 female Aedes mosquitoes were collected, of which 99.8% were Aedes aegypti and 0.2% were Aedes albopictus. Mean female Aedes mosquito density per household was 1.71 (standard deviation: 2.84). We used mixed-effects generalized linear Poisson regression analyses to identify predictors of Aedes density, using month, neighborhood and country as random-effects variables. Across study sites, the number of household occupants [incidence rate ratio (IRR): 1.08, 95% confidence interval (CI): 1.01–1.14], presence of entry points for mosquitoes into the household (IRR: 1.51, 95% CI: 1.30–1.76) and presence of decorative vegetation (IRR: 1.52, 95% CI: 1.22–1.88) were associated with higher Aedes density; while being in the highest wealth tertile of household wealth (IRR: 0.78, 95% CI: 0.66–0.92), knowledge of how arboviruses are transmitted (IRR: 0.94, 95% CI: 0.89–1.00) and regular emptying of water containers by occupants (IRR: 0.79, 95% CI: 0.67–0.92) were associated with lower Aedes density. Conclusions Our study addresses the complexities of arbovirus vectors of global significance at the interface between human and mosquito populations. Our results point to several predictors of Aedes mosquito vector density in countries with co-circulation of multiple Aedes-borne viruses, and point to modifiable risk factors that may be useful for disease prevention and control. Graphical Abstract

Background The global impact of Zika virus in Latin America and the Caribbean has drawn renewed attention to circulating mosquito-borne viruses in this region, such as dengue and chikungunya. Our objective was to assess socio-ecological factors associated with Aedes mosquito vector density as a measure of arboviral transmission risk in three cities of potential recent Zika virus introduction: Ibagué, Colombia; Manta, Ecuador; and Posadas, Argentina, in order to inform disease mitigation strategies. Methods We monitored Aedes mosquito populations over 12 months starting in 2018 in a total of 1,086 randomly selected households, using indoor and peridomestic mosquito collection methods. For each sampled household, we collected socio-economic data using structured questionnaires and data on microenvironmental conditions using iButton data loggers. Results A total of 3,230 female Aedes mosquitoes were collected, of which 99.8% were Ae. aegypti and 0.2% were Ae. albopictus. Mean female Aedes spp. mosquito abundance per household was 1.71. We modeled the interaction between neighborhood and household wealth profiles, and found significantly lower Aedes density in highest compared to lowest and middle wealth households, although the effect differed by level of neighborhood wealth. We used mixed-effects Poisson regression analyses to identify predictors of Aedes density, using month, neighborhood, and country as random-effect variables. Across study sites, the number of household occupants (Incidence rate ratio, IRR = 1.08), presence of entry points for mosquitoes into the household (IRR = 1.51), and presence of decorative vegetation (IRR = 1.52) were associated with higher Aedes density; while being in the highest wealth tertile of household wealth (IRR = 0.78), knowledge of how arboviruses are transmitted (IRR = 0.94) and regular emptying of water containers by occupants (IRR = 0.79) were associated with lower Aedes density. Conclusions Our study addresses the complexities of arbovirus vectors of global significance at the interface between society and the environment. Our results point to several determinants of Aedes mosquito vector density in countries with co-circulation of multiple Aedes-borne viruses, and could help us identify targets of practical intervention for disease prevention and control.

La finca agroecológica de Zamorano está en el valle de Yeguare, San Antonio de Oriente, Departamento de Francisco Morazán, Honduras, es comanejada por la Escuela Agrícola Panamericana, Zamorano. Esta finca tiene como propósito reproducir un sistema agroecológico que garantice su estructura, funcionamiento y biodiversidad. El objetivo de este estudio fue determinar su composición florística e identificar las especies de importancia ecológica y económica, así como su estado de conservación. Se identificaron 210 especies que pertenecen a 53 familias, las más comunes son Asteraceae, Fabaceae y Malvaceae. El 86% de las especies registradas son nativas, en su mayoría hierbas y árboles de importancia económica. Las especies más importantes son: Guazuma ulmifolia, Quercus oleoides, Luehea candida, Bursera simaruba, Mimosa tenuiflora, Verbesina punctata, Malvaviscus arboreus. Solo ocho especies se encuentran en la categoría de atención menor, según la lista roja de la Unión Internacional para la Conservación de la Naturaleza (UICN), y dos especies de orquídeas en la lista CITES. La finca agroecológica de Zamorano ofrece un potencial económico por las especies de uso medicinal, maderable y comestible. Además, este estudio florístico se considera una herramienta para la elaboración del plan de manejo de la finca.

Giant tailed bacterial viruses, or phages, such as Pseudomonas aeruginosa phage ϕKZ, have long genomes packaged into large, atypical virions. Many aspects of ϕKZ and related phage biology are poorly understood, mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240-kb SPN3US genome shares a core set of 91 genes with ϕKZ and related phages, ∼61 of which are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of 18 mutants enabled assignment of 13 genes as essential, information which could not have been determined by sequence-based searches for 11 genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 provided new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241 , which encodes an RNA polymerase β subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a “missing” β′ subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host interactions, and evolution. IMPORTANCE In recent years, there has been a paradigm shift in virology with the realization that extremely large viruses infecting prokaryotes (giant phages) can be found in many environments. A group of phages related to the prototype giant phage ϕKZ are of great interest due to their virions being among the most complex of prokaryotic viruses and their potential for biocontrol and phage therapy applications. Our understanding of the biology of these phages is limited, as a large proportion of their proteins have not been characterized and/or have been deemed putative without any experimental verification. In this study, we analyzed Salmonella phage SPN3US using a combination of genomics, genetics, and proteomics and in doing so revealed new information regarding giant phage head structure and assembly and virion RNA polymerase composition. Our findings demonstrate the suitability of SPN3US as a model phage for the growing group of phages related to ϕKZ.