Intracellular Diversity of WNV within Circulating Avian Peripheral Blood Mononuclear Cells Reveals Host-Dependent Patterns of Polyinfection.

Arthropod-borne virus (arbovirus) populations exist as mutant swarms that are maintained between arthropods and vertebrates. West Nile virus (WNV) population dynamics are host-dependent. In American crows, purifying selection is weak and population diversity is high compared to American robins, which have 100- to 1000-fold lower viremia. WNV passed in robins leads to fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows for higher genetic diversity within individual avian peripheral blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a molecularly barcoded WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes in each. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to the maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation.

The Incompetence of Mosquitoes-Can Zika Virus Be Adapted To Infect Culex tarsalis Cells?

The molecular evolutionary mechanisms underpinning virus-host interactions are increasingly recognized as key drivers of virus emergence, host specificity, and the likelihood that viruses can undergo a host shift that alters epidemiology and transmission biology. Zika virus (ZIKV) is mainly transmitted between humans by Aedes aegypti mosquitoes. However, the 2015 to 2017 outbreak stimulated discussion regarding the role of Culex spp. mosquitoes in transmission. Reports of ZIKV-infected Culex mosquitoes, in nature and under laboratory conditions, resulted in public and scientific confusion. We previously found that Puerto Rican ZIKV does not infect colonized Culex quinquefasciatus, Culex pipiens, or Culex tarsalis, but some studies suggest they may be competent ZIKV vectors. Therefore, we attempted to adapt ZIKV to Cx. tarsalis by serially passaging virus on cocultured Ae. aegypti (Aag2) and Cx. tarsalis (CT) cells to identify viral determinants of species specificity. Increasing fractions of CT cells resulted in decreased overall virus titer and no enhancement of Culex cell or mosquito infection. Next-generation sequencing of cocultured virus passages revealed synonymous and nonsynonymous variants throughout the genome that arose as CT cell fractions increased. We generated nine recombinant ZIKVs containing combinations of the variants of interest. None of these viruses showed increased infection of Culex cells or mosquitoes, demonstrating that variants associated with passaging were not specific to increased Culex infection. These results reveal the challenge of a virus adapting to a new host, even when pushed to adapt artificially. Importantly, they also demonstrate that while ZIKV may occasionally infect Culex mosquitoes, Aedes mosquitoes likely drive transmission and human risk. IMPORTANCE ZIKV is mainly transmitted between humans by Aedes mosquitoes. In nature, ZIKV-infected Culex mosquitoes have been found, and ZIKV infrequently infects Culex mosquitoes under laboratory conditions. Yet, most studies show that Culex mosquitoes are not competent vectors for ZIKV. We attempted to adapt ZIKV to Culex cells to identify viral determinants of species specificity. We sequenced ZIKV after it was passaged on a mixture of Aedes and Culex cells and found that it acquired many variants. We generated recombinant viruses containing combinations of the variants of interest to determine if any of these changes enhance infection in Culex cells or mosquitoes. Recombinant viruses did not show increased infection in Culex cells or mosquitoes, but some variants increased infection in Aedes cells, suggesting adaptation to those cells instead. These results reveal that arbovirus species specificity is complex, and that virus adaptation to a new genus of mosquito vectors likely requires multiple genetic changes.

Intracellular diversity of WNV within circulating avian peripheral blood mononuclear cells reveals host-dependent patterns of polyinfection.

Error-prone replication of RNA viruses generates the genetic diversity required for adaptation within rapidly changing environments. Thus, arthropod-borne virus (arbovirus) populations exist in nature as mutant swarms that are maintained between arthropods and vertebrates. Previous studies have demonstrated that West Nile virus (WNV) population dynamics are host dependent: In American crows, which experience extremely high viremia, purifying selection is weak and population diversity is high compared to American robins, which have 100 to 1000-fold lower viremia. WNV passed in robins experiences fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows higher genetic diversity within individual avian peripheral-blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a novel, barcoded version of WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes that each contained. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation. These studies further document the role of particular, ecologically relevant hosts in shaping virus population structure. Author Summary: WNV mutational diversity in vertebrates is species-dependent. In crows, low frequency variants are common, and viral populations are more diverse. In robins, fewer mutations become permanent fixtures of the overall viral population. We infected crows, robins and a chicken cell line with a genetically marked (barcoded) WNV. Higher levels of virus led to multiple unique WNV genomes infecting individual cells, even when a genotype was present at low levels in the input viral stock. Our findings suggest that higher levels of circulating virus in natural hosts allow less fit viruses to survive in RNA virus populations through complementation by more fit viruses. This is significant as it allows less represented and less fit viruses to be maintained at low levels until they potentially emerge when virus environments change. Overall our data reveal new insights on the relationships between host susceptibility to high viremia and virus evolution.

Impact of extrinsic incubation temperature on natural selection during Zika virus infection of Aedes aegypti and Aedes albopictus.

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes.

Infection, dissemination, and transmission efficiencies of Zika virus in Aedes aegypti after serial passage in mosquito or mammalian cell lines or alternating passage in both cell types.

BACKGROUND: Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) with an urban transmission cycle that primarily involves humans and Aedes aegypti. Evidence suggests that the evolution of some arboviruses is constrained by their dependency on alternating between disparate (vertebrate and invertebrate) hosts. The goals of this study are to compare the genetic changes that occur in ZIKV after serial passaging in mosquito or vertebrate cell lines or alternate passaging in both cell types and to compare the replication, dissemination, and transmission efficiencies of the cell culture-derived viruses in Ae. aegypti. METHODS: An isolate of ZIKV originally acquired from a febrile patient in Yucatan, Mexico, was serially passaged six times in African green monkey kidney (Vero) cells or Aedes albopictus (C6/36) cells or both cell types by alternating passage. A colony of Ae. aegypti from Yucatan was established, and mosquitoes were challenged with the cell-adapted viruses. Midguts, Malpighian tubules, ovaries, salivary glands, wings/legs and saliva were collected at various times after challenge and tested for evidence of virus infection. RESULTS: Genome sequencing revealed the presence of two non-synonymous substitutions in the premembrane and NS1 regions of the mosquito cell-adapted virus and two non-synonymous substitutions in the capsid and NS2A regions of both the vertebrate cell-adapted and alternate-passaged viruses. Additional genetic changes were identified by intrahost variant frequency analysis. Virus maintained by continuous C6/36 cell passage was significantly more infectious in Ae. aegypti than viruses maintained by alternating passage and consecutive Vero cell passage. CONCLUSIONS: Mosquito cell-adapted ZIKV displayed greater in vivo fitness in Ae. aegypti compared to the other viruses, indicating that obligate cycling between disparate hosts carries a fitness cost. These data increase our understanding of the factors that drive ZIKV adaptation and evolution and underscore the important need to consider the in vivo passage histories of flaviviruses to be evaluated in vector competence studies.

Genome Number and Size Polymorphism in Zika Virus Infectious Units.

Zika virus (ZIKV; Flaviviridae, Flavivirus) is an arthropod-borne infection that can result in severe outcomes, particularly in fetuses infected in utero It has been assumed that infection by ZIKV, as well as other viruses, is largely initiated by individual virus particles binding to and entering a cell. However, recent studies have demonstrated that multiple virus particles are frequently delivered to a cell simultaneously and that this collective particle delivery enhances infection. ZIKV is maintained in nature between Aedes aegypti mosquitos and vertebrate hosts, including humans. Human infection is initiated through the injection of a relatively small initial inoculum comprised of a genetically complex virus population. Since most mutations decrease virus fitness, collective particle transmission could benefit ZIKV and other arthropod-borne diseases by facilitating the maintenance of genetic complexity and adaptability during infection or through other mechanisms. Therefore, we utilized a barcoded ZIKV to quantify the number of virus genomes that initiate a plaque. We found that individual plaques contain a mean of 10 infecting viral genomes (range, 1 to 212). Few plaques contained more than two dominant genomes. To determine whether multigenome infectious units consist of collectively transmitting virions, infectious units of ZIKV were then separated mechanically by centrifugation, and heavier fractions were found to contain more genomes per plaque-forming unit, with larger diameters. Finally, larger/heavier infectious units reformed after removal. These data suggest that ZIKV populations consist of a variety of infectious unit sizes, likely mostly made up of aggregates, and only rarely begin with a single virus genome.IMPORTANCE The arthropod-borne Zika virus (ZIKV) infects humans and can cause severe neurological sequelae, particularly in fetuses infected in utero How this virus has been able to spread across vast geological ranges and evolve in new host populations is not yet understood. This research demonstrates a novel mechanism of ZIKV transmission through multigenome aggregates, providing insight into ZIKV evolution, immunologic evasion, and better future therapeutic design. This study shows that ZIKV plaques result from collections of genomes rather than individual genomes, increasing the potential for interactions between ZIKV genotypes.

Bat influenza viruses transmit among bats but are poorly adapted to non-bat species.

Major histocompatibility complex class II (MHC-II) molecules of multiple species function as cell-entry receptors for the haemagglutinin-like H18 protein of the bat H18N11 influenza A virus, enabling tropism of the viruses in a potentially broad range of vertebrates. However, the function of the neuraminidase-like N11 protein is unknown because it is dispensable for viral infection or the release of H18-pseudotyped viruses. Here, we show that infection of mammalian cells with wild-type H18N11 leads to the emergence of mutant viruses that lack the N11 ectodomain and acquired mutations in H18. An infectious clone of one such mutant virus, designated rP11, appeared to be genetically stable in mice and replicated to higher titres in mice and cell culture compared with wild-type H18N11. In ferrets, rP11 antigen and RNA were detected at low levels in various tissues, including the tonsils, whereas the wild-type virus was not. In Neotropical Jamaican fruit bats, wild-type H18N11 was found in intestinal Peyer's patches and was shed to high concentrations in rectal samples, resulting in viral transmission to naive contact bats. Notably, rP11 also replicated efficiently in bats; however, only restored full-length N11 viruses were transmissible. Our findings suggest that wild-type H18N11 replicates poorly in mice and ferrets and that N11 is a determinant for viral transmission in bats.

Zika viruses of African and Asian lineages cause fetal harm in a mouse model of vertical transmission.

Congenital Zika virus (ZIKV) infection was first linked to birth defects during the American outbreak in 2015/2016. It has been proposed that mutations unique to the Asian/American-genotype explain, at least in part, the ability of Asian/American ZIKV to cause congenital Zika syndrome (CZS). Recent studies identified mutations in ZIKV infecting humans that arose coincident with the outbreak in French Polynesia and were stably maintained during subsequent spread to the Americas. Here we show that African ZIKV can infect and harm fetuses and that the S139N substitution that has been associated with the American outbreak is not essential for fetal harm. Our findings, in a vertical transmission mouse model, suggest that ZIKV will remain a threat to pregnant women for the foreseeable future, including in Africa, Southeast Asia, and the Americas. Additional research is needed to better understand the risks associated with ZIKV infection during pregnancy, both in areas where the virus is newly endemic and where it has been circulating for decades.

Variation in competence for ZIKV transmission by Aedes aegypti and Aedes albopictus in Mexico.

BACKGROUND: ZIKV is a new addition to the arboviruses circulating in the New World, with more than 1 million cases since its introduction in 2015. A growing number of studies have reported vector competence (VC) of Aedes mosquitoes from several areas of the world for ZIKV transmission. Some studies have used New World mosquitoes from disparate regions and concluded that these have a variable but relatively low competence for the Asian lineage of ZIKV. METHODOLOGY/PRINCIPAL FINDINGS: Ten Aedes aegypti (L) and three Ae. albopictus (Skuse) collections made in 2016 from throughout Mexico were analyzed for ZIKV (PRVABC59-Asian lineage) VC. Mexican Ae. aegypti had high rates of midgut infection (MIR), dissemination (DIR) and salivary gland infection (SGIR) but low to moderate transmission rates (TR). It is unclear whether this low TR was due to heritable salivary gland escape barriers or to underestimating the amount of virus in saliva due to the loss of virus during filtering and random losses on surfaces when working with small volumes. VC varied among collections, geographic regions and whether the collection was made north or south of the Neovolcanic axis (NVA). The four rates were consistently lower in northeastern Mexico, highest in collections along the Pacific coast and intermediate in the Yucatan. All rates were lowest north of the NVA. It was difficult to assess VC in Ae. albopictus because rates varied depending upon the number of generations in the laboratory. CONCLUSIONS/SIGNIFICANCE: Mexican Ae. aegypti and Ae. albopictus are competent vectors of ZIKV. There is however large variance in vector competence among geographic sites and regions. At 14 days post infection, TR varied from 8-51% in Ae. aegypti and from 2-26% in Ae. albopictus.

American Aedes vexans Mosquitoes are Competent Vectors of Zika Virus.

AbstractStarting in 2013-2014, the Americas have experienced a massive outbreak of Zika virus (ZIKV) which has now reached at least 49 countries. Although most cases have occurred in South America and the Caribbean, imported and autochthonous cases have occurred in the United States. Aedes aegypti and Aedes albopictus mosquitoes are known vectors of ZIKV. Little is known about the potential for temperate Aedes mosquitoes to transmit ZIKV. Aedes vexans has a worldwide distribution, is highly abundant in particular localities, aggressively bites humans, and is a competent vector of several arboviruses. However, it is not clear whether Ae. vexans mosquitoes are competent to transmit ZIKV. To determine the vector competence of Ae. vexans for ZIKV, wild-caught mosquitoes were exposed to an infectious bloodmeal containing a ZIKV strain isolated during the current outbreak. Approximately 80% of 148 mosquitoes tested became infected by ZIKV, and approximately 5% transmitted infectious virus after 14 days of extrinsic incubation. These results establish that Ae. vexans are competent ZIKV vectors. Their relative importance as vectors (i.e., their vectorial capacity) depends on feeding behavior, longevity, and other factors that are likely to vary in ecologically distinct environments.

Impact of simultaneous exposure to arboviruses on infection and transmission by Aedes aegypti mosquitoes.

The recent emergence of both chikungunya and Zika viruses in the Americas has significantly expanded their distribution and has thus increased the possibility that individuals may become infected by more than one Aedes aegypti-borne virus at a time. Recent clinical data support an increase in the frequency of coinfection in human patients, raising the likelihood that mosquitoes could be exposed to multiple arboviruses during one feeding episode. The impact of coinfection on the ability of relevant vector species to transmit any of these viruses (that is, their vector competence) has not been determined. Thus, we here expose Ae. aegypti mosquitoes to chikungunya, dengue-2 or Zika viruses, both individually and as double and triple infections. Our results show that these mosquitoes can be infected with and can transmit all combinations of these viruses simultaneously. Importantly, infection, dissemination and transmission rates in mosquitoes are only mildly affected by coinfection.

Mosquitoes Transmit Unique West Nile Virus Populations during Each Feeding Episode.

Arthropod-borne viruses (arboviruses), such as Zika virus, chikungunya virus, and West Nile virus (WNV), pose continuous threats to emerge and cause large epidemics. Often, these events are associated with novel virus variants optimized for local transmission that first arise as minorities within a host. Thus, the conditions that regulate the frequency of intrahost variants are important determinants of emergence. Here, we describe the dynamics of WNV genetic diversity during its transmission cycle. By temporally sampling saliva from individual mosquitoes, we demonstrate that virus populations expectorated by mosquitoes are highly diverse and unique to each feeding episode. After transmission to birds, however, most genetic diversity is removed by strong purifying selection. Further, transmission of potentially mosquito-adaptive WNV variants is strongly influenced by genetic drift in mosquitoes. These results highlight the complex evolutionary forces a novel virus variant must overcome to alter infection phenotypes at the population level.

Genetic Drift during Systemic Arbovirus Infection of Mosquito Vectors Leads to Decreased Relative Fitness during Host Switching.

The emergence of mosquito-borne RNA viruses, such as West Nile virus (WNV), is facilitated by genetically complex virus populations within hosts. Here, we determine whether WNV enzootic (Culex tarsalis, Cx. quinquefasciatus, and Cx. pipiens) and bridge vectors (Aedes aegypti) have differential impacts on viral mutational diversity and fitness. During systemic mosquito infection, WNV faced stochastic reductions in genetic diversity that rapidly was recovered during intra-tissue population expansions. Interestingly, this intrahost selection and diversification was mosquito species dependent with Cx. tarsalis and Cx. quinquefasciatus exhibiting greater WNV divergence. However, recovered viral populations contained a preponderance of potentially deleterious mutations (i.e., high mutational load) and had lower relative fitness in avian cells compared to input virus. These findings demonstrate that the adaptive potential associated with mosquito transmission varies depending on the mosquito species and carries a significant fitness cost in vertebrates.