Like other arthropod-borne viruses (arboviruses), mosquito-borne dengue virus (DENV) is maintained in an alternating cycle of replication in arthropod and vertebrate hosts. The trade-off hypothesis suggests that this alternation constrains DENV evolution because a fitness increase in one host usually diminishes fitness in the other. Moreover, the hypothesis predicts that releasing DENV from host alternation should facilitate adaptation. To test this prediction, DENV was serially passaged in either a single human cell line (Huh-7), a single mosquito cell line (C6/36), or in alternating passages between Huh-7 and C6/36 cells. After 10 passages, consensus mutations were identified and fitness was assayed by evaluating replication kinetics in both cell types as well as in a novel cell type (Vero) that was not utilized in any of the passage series. Viruses allowed to specialize in single host cell types exhibited fitness gains in the cell type in which they were passaged, but fitness losses in the bypassed cell type, and most alternating passages, exhibited fitness gains in both cell types. Interestingly, fitness gains were observed in the alternately passaged, cloned viruses, an observation that may be attributed to the acquisition of both host cell–specific and amphi-cell-specific adaptations or to recovery from the fitness losses due to the genetic bottleneck of biological cloning. Amino acid changes common to both passage series suggested convergent evolution to replication in cell culture via positive selection. However, intriguingly, mutations accumulated more rapidly in viruses passed in Huh-7 cells than in those passed in C6/36 cells or in alternation. These results support the hypothesis that releasing DENV from host alternation facilitates adaptation, but there is limited support for the hypothesis that such alternation necessitates a fitness trade-off. Moreover, these findings suggest that patterns of genetic evolution may differ between viruses replicating in mammalian and mosquito cells.

Mosquito-borne alphaviruses, which replicate alternately and obligately in mosquitoes and vertebrates, appear to experience lower rates of evolution than do many RNA viruses that replicate solely in vertebrates. This genetic stability is hypothesized to result from the alternating host cycle, which constrains evolution by imposing compromise fitness solutions in each host. To test this hypothesis, Sindbis virus was passaged serially, either in one cell type to eliminate host alteration or alternately between vertebrate (BHK) and mosquito (C6/36) cells. Following 20 to 50 serial passages, mutations were identified and changes in fitness were assessed using competition assays against genetically marked, surrogate parent viruses. Specialized viruses passaged in a single cell exhibited more mutations and amino acid changes per passage than those passaged alternately. Single host-adapted viruses exhibited fitness gains in the cells in which they specialized but fitness losses in the bypassed cell type. Most but not all viruses passaged alternately experienced lesser fitness gains than specialized viruses, with fewer mutations per passage. Clonal populations derived from alternately passaged viruses also exhibited adaptation to both cell lines, indicating that polymorphic populations are not required for simultaneous fitness gains in vertebrate and mosquito cells. Nearly all passaged viruses acquired Arg or Lys substitutions in the E2 envelope glycoprotein, but enhanced binding was only detected for BHK cells. These results support the hypothesis that arbovirus evolution may be constrained by alternating host transmission cycles, but they indicate a surprising ability for simultaneous adaptation to highly divergent cell types by combinations of mutations in single genomes.Sindbis virus (SINV) is the type species of the genus Alphavirus, a group of RNA viruses with nonsegmented singlestranded genomes of approximately 11.7 kb (23, 25). The alphavirus genome is capped at the 5Ј end and contains a 3Ј poly(A) tail. The 5Ј two-thirds of the genome encode the nonstructural proteins 1 to 4, which are necessary for viral replication. The structural proteins, capsid and E1 and E2 envelope glycoproteins, are translated from a subgenomic mRNA (26S) which is identical in sequence to the 3Ј one-third of the genome (Fig. 1). The Sindbis virion contains an icosahedral nucleocapsid that consists of 240 copies of the capsid protein surrounded by a lipid envelope derived from the plasma membrane of infected cells, into which the glycoprotein E1/E2 heterodimers are embedded (4).Nearly all alphaviruses rely on horizontal mosquito-borne transmission among vertebrate hosts, requiring alternating replication in highly divergent hosts and cell types (7,34). Alphaviruses and other arthropod-borne viruses also appear to undergo lower rates of evolution than many other animal RNA viruses that replicate solely in vertebrates (6,33,35). Rates of SINV nucleotide substitution, deduced from oligonucleotide fingerprinting, are approximately 4 ϫ 10 Ϫ4 ...

In nature, arthropod-borne viruses (arboviruses) perpetuate through alternating replication in vertebrate and invertebrate hosts. The trade-off hypothesis proposes that these viruses maintain adequate replicative fitness in two disparate hosts in exchange for superior fitness in one host. Releasing the virus from the constraints of a two-host cycle should thus facilitate adaptation to a single host. This theory has been addressed in a variety of systems, but remains poorly understood. We sought to determine the fitness implications of alternating host replication for West Nile virus (WNV) using an in vivo model system. Previously, WNV was serially or alternately passed 20 times in vivo in chicks or mosquitoes and resulting viruses were characterized genetically. In this study, these test viruses were competed in vivo in fitness assays against an unpassed marked reference virus. Fitness was assayed in chicks and in two important WNV vectors, Culex pipiens and Culex quinquefasciatus. Chick-specialized virus displayed clear fitness gains in chicks and in Cx. pipiens but not in Cx. quinquefasciatus. Cx. pipiens-specialized virus experienced reduced fitness in chicks and little change in either mosquito species. These data suggest that when fitness is measured in birds the trade-off hypothesis is supported; but in mosquitoes it is not. Overall, these results suggest that WNV evolution is driven by alternate cycles of genetic expansion in mosquitoes, where purifying selection is weak and genetic diversity generated, and restriction in birds, where purifying selection is strong.

Specimens and associated data in natural history collections (NHCs) foster substantial scientific progress. In this paper, we explore recent contributions of NHCs to the study of systematics and biogeography, genomics, morphology, stable isotope ecology, and parasites and pathogens of mammals. To begin to assess the magnitude and scope of these contributions, we analyzed publications in the over the last decade, as well as recent research supported by a single university mammal collection (Museum of Southwestern Biology, Division of Mammals). Using these datasets, we also identify weak links that may be hindering the development of crucial NHC infrastructure. Maintaining the vitality and growth of this foundation of mammalogy depends on broader engagement and support from across the scientific community and is both an ethical and scientific imperative given the rapidly changing environmental conditions on our planet.

Complete genome sequencing of 22 West Nile virus isolates suggested 2 independent introductions into Mexico. A previously identified mouse-attenuated glycosylation variant was introduced into southern Mexico through the southeastern United States, while a common US genotype appears to have been introduced incrementally into northern Mexico through the southwestern United States.