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. Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.

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.

Following the recent discovery of Bourbon virus (BRBV) as a human pathogen, and the isolation of the virus from Amblyomma americanum (L.) collected near the location of a fatal human case, we undertook a series of experiments to assess the laboratory vector competence of this tick species for BRBV. Larval ticks were infected using an immersion technique, and transstadial transmission of virus to the nymphal and then to the adult stages was demonstrated. Transstadially infected nymphs transmitted virus to adult ticks at very high rates during cofeeding, indicating the presence of infectious virus in the saliva of engorging ticks. Vertical transmission by transstadially infected females to their progeny occurred, but at a low rate. Rabbits fed on by infected ticks of all active life stages developed high titers of antibody to the virus, demonstrating host exposure to BRBV antigens/live virus during tick blood feeding. These results demonstrate that A. americanum is a competent vector of BRBV and indicate that cofeeding could be critical for enzootic maintenance.

Since its recent discovery, Bourbon virus has been isolated from a human and ticks. To assess exposure of potential vertebrate reservoirs, we assayed banked serum and plasma samples from wildlife and domestic animals in Missouri, USA, for Bourbon virus-neutralizing antibodies. We detected high seroprevalence in raccoons (50%) and white-tailed deer (86%).

West Nile virus (WNV) and St. Louis encephalitis (SLEV) virus are enzootically maintained in North America in cycles involving the same mosquito vectors and similar avian hosts. However, these viruses exhibit dissimilar viremia and virulence phenotypes in birds: WNV is associated with high magnitude viremias that can result in mortality in certain species such as American crows (AMCRs, Corvus brachyrhynchos) whereas SLEV infection yields lower viremias that have not been associated with avian mortality. Cross-neutralization of these viruses in avian sera has been proposed to explain the reduced circulation of SLEV since the introduction of WNV in North America; however, in 2015, both viruses were the etiologic agents of concurrent human encephalitis outbreaks in Arizona, indicating the need to re-evaluate host factors and cross-neutralization responses as factors potentially affecting viral co-circulation. Reciprocal chimeric WNV and SLEV viruses were constructed by interchanging the pre-membrane (prM)-envelope (E) genes, and viruses subsequently generated were utilized herein for the inoculation of three different avian species: house sparrows (HOSPs; Passer domesticus), house finches (Haemorhous mexicanus) and AMCRs. Cross-protective immunity between parental and chimeric viruses were also assessed in HOSPs. Results indicated that the prM-E genes did not modulate avian replication or virulence differences between WNV and SLEV in any of the three avian species. However, WNV-prME proteins did dictate cross-protective immunity between these antigenically heterologous viruses. Our data provides further evidence of the important role that the WNV / SLEV viral non-structural genetic elements play in viral replication, avian host competence and virulence.

Heartland virus (HRTV) is a recently described phlebovirus initially isolated in 2009 from 2 humans who had leukopenia and thrombocytopenia. Serologic assessment of domestic and wild animal populations near the residence of 1 of these persons showed high exposure rates to raccoons, white-tailed deer, and horses. To our knowledge, no laboratory-based assessments of viremic potential of animals infected with HRTV have been performed. We experimentally inoculated several vertebrates (raccoons, goats, chickens, rabbits, hamsters, C57BL/6 mice, and interferon-alpha/beta/gamma receptor-deficient [Ag129]) mice with this virus. All animals showed immune responses against HRTV after primary or secondary exposure. However, neutralizing antibody responses were limited. Only Ag129 mice showed detectable viremia and associated illness and death, which were dose dependent. Ag129 mice also showed development of mean peak viral antibody titers >8 log10 PFU/mL, hemorrhagic hepatic lesions, splenomegaly, and large amounts of HRTV antigen in mononuclear cells and hematopoietic cells in the spleen.

West Nile virus (WNV) is an important agent of human encephalitis that has quickly become endemic across much of the United States since its identification in North America in 1999. While the majority ( approximately 75%) of infections are subclinical, neurologic disease can occur in a subset of cases, with outcomes including permanent neurologic damage and death. Currently, there are no WNV vaccines approved for use in humans. This study introduces a novel vaccine platform for WNV to reduce viral replication in the central nervous system while maintaining peripheral replication to elicit strong neutralizing antibody titers. Vaccine candidates were engineered to incorporate microRNA (miRNA) target sequences for a cognate miRNA expressed only in neurons, allowing the host miRNAs to target viral transcription through endogenous RNA silencing. To maintain stability, these targets were incorporated in multiple locations within the 3'-untranslated region, flanking sequences essential for viral replication without affecting the viral open reading frame. All candidates replicated comparably to wild type WNV in vitro within cells that did not express the cognate miRNA. Insertional control viruses were also capable of neuroinvasion and neurovirulence in vivo in CD-1 mice. Vaccine viruses were safe at all doses tested and did not demonstrate mutations associated with a reversion to virulence when serially passaged in mice. All vaccine constructs were protective from lethal challenge in mice, producing 93-100% protection at the highest dose tested. Overall, this is a safe and effective attenuation strategy with broad potential application for vaccine development.

Heartland virus (HRTV; Bunyaviridae: Phlebovirus) is a recently described cause of human illness in the United States. After field studies conducted in 2012 implicated Amblyomma americanum (L.) as a vector of HRTV, we initiated experiments to assess the vector competence of A. americanum Larval and nymphal ticks were immersed in high-titered suspensions of HRTV, and then tested for virus at various intervals postimmersion. In a later trial larval ticks were immersed in HRTV, followed by engorgement on a rabbit. A subset of postmolt nymphs was tested for HRTV to document transstadial transmission. Putatively infected nymphs were cofed with uninfected colony larvae to assess nonviremic transmission. In another trial, nymphs were fed on a rabbit and allowed to molt to the adult stage. Male and female ticks fed and mated upon a rabbit, and females were allowed to oviposit. Male and spent female ticks were tested for HRTV, and offspring of infected females were tested to assess vertical transmission. Infection rates of ≤50% were observed in immersed larvae and nymphs tested at intervals following immersion. Transstadial transmission from larvae to nymphs and then to adults was documented. HRTV was detected in a pool of nymphs molted from uninfected larvae cofed with infected nymphs. Vertical transmission of HRTV was observed in progeny of infected females. Infected females took longer to oviposit and produced fewer offspring. Serologic conversions (without viremia) in rabbits fed upon by immersed larvae or transstadially infected ticks indicate horizontal transmission of HRTV.

BACKGROUND: In June of 2014, a previously healthy man from Kansas with a recent history of tick exposure died from complications related to an illness marked by fever, thrombocytopenia and leukopenia. An isolate was derived from the blood of this patient during the course of diagnostic testing. This isolate was subsequently identified as a novel orthomyxovirus of the genus Thogotovirus by next generation sequencing and was named Bourbon virus after the patient's county of residence. OBJECTIVES: To support research and diagnostic aims, we provide a basic description of Bourbon virus at both the molecular and serological levels. Furthermore, to preliminarily identify potential host and vector range associations we have characterized the growth kinetics of Bourbon virus in a variety of vertebrate and invertebrate cell lines. STUDY DESIGN: Bourbon virus was subjected to next generation-high throughput sequencing, phylogenetic, and basic structural protein analyses as well as 2-way plaque reduction neutralization assays. Also, we inoculated a variety of cell types with Bourbon virus and evaluated the growth kinetics by determining viral titers in the supernatants taken from infected cells over time. RESULTS: Bourbon virus possesses 24-82% identity at the amino acid sequence level and low serological cross-reactivity with other Thogotoviruses. In vitro growth kinetics reveal robust replication of Bourbon virus in mammalian and tick cells. CONCLUSIONS: Molecular and serological characterizations identify Bourbon virus as a novel member of the genus Thogotovirus. Results from cell culture analyses suggest an association between Bourbon virus and mammalian and tick hosts.

West Nile virus (WNV) outbreaks in North America have been characterized by substantial die-offs of American crows (Corvus brachyrhynchos). In contrast, a low incidence of bird deaths has been observed during WNV epidemic activity in Europe. To examine the susceptibility of the western European counterpart of American crows, we inoculated carrion crows (Corvus corone) with WNV strains isolated in Greece (Gr-10), Italy (FIN and Ita09), and Hungary (578/10) and with the highly virulent North American genotype strain (NY99). We also inoculated American crows with a selection of these strains to examine the strains' virulence in a highly susceptible bird species. Infection with all strains, except WNV FIN, resulted in high rates of death and high-level viremia in both bird species and virus dissemination to several organs. These results suggest that carrion crows are highly susceptible to WNV and may potentially be useful as part of dead bird surveillance for early warning of WNV activity in Europe.

Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7x more than sparrows, ~3.4x more than robins) and defective genomes (~1.4x greater than sparrows, ~2.7x greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6x lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses.

Heartland virus (HRTV; Bunyaviridae, Phlebovirus) has recently emerged as the causative agent of human disease characterized by thrombocytopenia and leukopenia in the United States. The lone star tick (Amblyomma americanum L.) has been implicated as a vector. To identify candidate vertebrate amplification hosts associated with enzootic maintenance of the virus, sera and ticks were sampled from 160 mammals (8 species) and 139 birds (26 species) captured near two human case residences in Andrew and Nodaway Counties in northwest Missouri. HRTV-specific neutralizing antibodies were identified in northern raccoons (42.6%), horses (17.4%), white-tailed deer (14.3%), dogs (7.7%), and Virginia opossums (3.8%), but not in birds. Virus isolation attempts from sera and ticks failed to detect HRTV virus. The high antibody prevalence coupled with local abundance of white-tailed deer and raccoons indicates these species as candidate amplification hosts.

West Nile virus (WNV) is a human pathogen of significant medical importance with close to 40,000 cases of encephalitis and more than 1,600 deaths reported in the US alone since its first emergence in New York in 1999. Previous studies identified a motif in the beginning of non-structural gene NS2A of encephalitic flaviviruses including WNV which induces programmed -1 ribosomal frameshift (PRF) resulting in production of an additional NS protein NS1'. We have previously demonstrated that mutant WNV with abolished PRF was attenuated in mice. Here we have extended our previous observations by showing that PRF does not appear to have a significant role in virus replication, virion formation, and viral spread in several cell lines in vitro. However, we have also shown that PRF induces an over production of structural proteins over non-structural proteins in virus-infected cells and that mutation abolishing PRF is present in ∼11% of the wild type virus population. In vivo experiments in house sparrows using wild type and PRF mutant of New York 99 strain of WNV viruses showed some attenuation for the PRF mutant virus. Moreover, PRF mutant of Kunjin strain of WNV showed significant decrease compared to wild type virus infection in dissemination of the virus from the midgut through the haemocoel, and ultimately the capacity of infected mosquitoes to transmit virus. Thus our results demonstrate an important role for PRF in regulating expression of viral genes and consequently virus replication in avian and mosquito hosts.

In July, 2012 three isolations were made from mosquitoes collected in Brazoria, Orange and Montgomery counties, Texas, USA. Data from immunofluorescence testing suggested that these isolates are members of the genus Orthobunyavirus. Expanded analyses confirmed that these isolates comprise three independent isolations of the same virus; a novel orthobunyavirus. The genetic organization of the M and L segments of this virus is similar to that of other orthobunyaviruses. However, the S segment ( approximately 1.7kb) is nearly twice the length of known orthobunyavirus S segments, encoding a significantly larger nucleocapsid, N ( approximately 50kDa) and putative non-structural NSs ( approximately 20kDa) proteins in a novel strategy by which the NSs ORF precedes the N ORF. The N protein appears to consist of two functional domains; an amino portion that possesses motifs similar to other orthobunyavirus N proteins and a carboxyl portion that possesses a glutamine-rich domain with no known homologue among Bunyaviridae.