Interspecies co-feeding transmission of Powassan virus between a native tick, Ixodes scapularis, and the invasive East Asian tick, Haemaphysalis longicornis.

BACKGROUND: Powassan virus, a North American tick-borne flavivirus, can cause severe neuroinvasive disease in humans. While Ixodes scapularis are the primary vectors of Powassan virus lineage II (POWV II), also known as deer tick virus, recent laboratory vector competence studies showed that other genera of ticks can horizontally and vertically transmit POWV II. One such tick is the Haemaphysalis longicornis, an invasive species from East Asia that recently established populations in the eastern USA and already shares overlapping geographic range with native vector species such as I. scapularis. Reports of invasive H. longicornis feeding concurrently with native I. scapularis on multiple sampled hosts highlight the potential for interspecies co-feeding transmission of POWV II. Given the absence of a clearly defined vertebrate reservoir host for POWV II, it is possible that this virus is sustained in transmission foci via nonviremic transmission between ticks co-feeding on the same vertebrate host. The objective of this study was to evaluate whether uninfected H. longicornis co-feeding in close proximity to POWV II-infected I. scapularis can acquire POWV independent of host viremia. METHODS: Using an in vivo tick transmission model, I. scapularis females infected with POWV II ("donors") were co-fed on mice with uninfected H. longicornis larvae and nymphs ("recipients"). The donor and recipient ticks were infested on mice in various sequences, and mouse infection status was monitored by temporal screening of blood for POWV II RNA via quantitative reverse transcription polymerase chain reaction (q-RT-PCR). RESULTS: The prevalence of POWV II RNA was highest in recipient H. longicornis that fed on viremic mice. However, nonviremic mice were also able to support co-feeding transmission of POWV, as demonstrated by the detection of viral RNA in multiple H. longicornis dispersed across different mice. Detection of viral RNA at the skin site of tick feeding but not at distal skin sites indicates that a localized skin infection facilitates transmission of POWV between donor and recipient ticks co-feeding in close proximity. CONCLUSIONS: This is the first report examining transmission of POWV between co-feeding ticks. Against the backdrop of multiple unknowns related to POWV ecology, findings from this study provide insight on possible mechanisms by which POWV could be maintained in nature.

Horizontal and Vertical Transmission of Powassan Virus by the Invasive Asian Longhorned Tick, Haemaphysalis longicornis, Under Laboratory Conditions.

The Asian longhorned tick, Haemaphysalis longicornis, is an ixodid tick native to East Asia that was first detected in North America outside a port of entry in 2017. This invasive species has since been detected in 17 states. As the invasive range of the tick continues to expand, the vector competence of H. longicornis for pathogens native to North America must be assessed. Here, we evaluate the vector competence of H. longicornis for Powassan virus (POWV) under laboratory conditions. POWV is a North American tick-borne flavivirus that is typically transmitted through the bite of Ixodes species ticks. The invasive range of H. longicornis is expected to overlap heavily with the geographic range of Ixodes scapularis and POWV cases, highlighting the potential for this invasive tick species to amplify POWV transmission in natural foci should the native tick vectors and H. longicornis share similar hosts. In these studies, adult female H. longicornis ticks were infected with POWV via anal pore microinjection. Viral RNA and infectious virions were detected in tick tissues via q-RT-PCR and focus-forming assay, respectively. POWV-injected female ticks were infested on mice, and virus was transmitted to mice during tick feeding, as shown by clinical signs of disease and seroconversion in the tick-exposed mice, as well as the detection of viral RNA in various mouse tissues. A POWV-injected female tick transmitted virus to her larval progeny, indicating that H. longicornis can vertically transmit POWV. These naturally-infected larval ticks were also able to transmit POWV to the mouse on which they fed and to the nymphal stage after molting, further demonstrating that H. longicornis can transmit POWV in the horizontal and transstadial modes. Larval and nymphal ticks were also orally infected with POWV while feeding on viremic mice. Additionally, this study provides the first report of POWV neuropathology based on a natural tick transmission model of POWV. Together, our results suggest that the invasive H. longicornis tick is a competent vector of POWV. These findings underline the growing danger this tick may pose to human health in the United States. Additional scholarship on the tick's biology, ecology, and pathogen transmission dynamics in nature will be important towards understanding the full public health impact of this invasive species.

Transovarial Transmission of Heartland Virus by Invasive Asian Longhorned Ticks under Laboratory Conditions.

We demonstrated experimental acquisition and transmission of Heartland bandavirus by Haemaphysalis longicornis ticks. Virus was detected in tick salivary gland and midgut tissues. A total of 80% of mice exposed to 1 infected tick seroconverted, suggesting horizontal transmission. H. longicornis ticks can transmit the virus in the transovarial mode.

Salivary gland extract from the deer tick, Ixodes scapularis, facilitates neuroinvasion by Powassan virus in BALB/c mice.

Powassan virus (POWV) is a neuroinvasive flavivirus transmitted to mammals by the bite of ixodid ticks. In this study, we sought to investigate the impact of tick salivary gland extract (SGE) on POWV neuroinvasion. BALB/c mice were footpad inoculated with either a high dose or a low dose of POWV, with and without Ixodes scapularis salivary gland extract. Brain and spinal cord were extracted daily, and immunohistochemical techniques were used for temporal tracking of POWV antigen. The temporal pattern of POWV staining showed a caudal to rostral spread of POWV in the brains of mice from both high dose infection groups. For the high dose infection groups, the presence of tick SGE did not influence the spread of POWV in the brain. Mice infected with the low dose of virus alone did not present POWV staining in the brain; however, in the presence of SGE, low dose infected mice presented scattered foci of POWV-infected cells throughout the brain. This study shows that tick SGE facilitates POWV neuroinvasion when mice are infected with the lower dose of POWV. We also found two patterns of central nervous system invasion that were directly influenced by the dose of POWV administered.

Development of a small animal model for deer tick virus pathogenesis mimicking human clinical outcome.

Powassan virus (POWV) is a tick-borne flavivirus that encompasses two genetic lineages, POWV (Lineage I) and deer tick virus (DTV, Lineage II). In recent years, the incidence of reported POWV disease cases has increased, coupled with an expanded geographic range of the DTV tick vector, Ixodes scapularis. POWV and DTV are serologically indistinguishable, and it is not known whether clinical manifestations, pathology, or disease outcome differ between the two viruses. Six-week-old male and female BALB/c mice were footpad-inoculated with DTV doses ranging from 101 to 105 FFU. Dose-independent mortality, morbidity, and organ viral loads were observed for mice inoculated with sequentially increasing doses of DTV. By study completion, all surviving mice had cleared their viremias but detectable levels of negative-sense DTV RNA were present in the brain, indicating viral persistence of infectious DTV in the central nervous system. For mice that succumbed to disease, neuropathology revealed meningoencephalitis characterized by microscopic lesions with widespread distribution of viral RNA in the brain. These findings, coupled with the rapid onset of neurological signs of disease and high viral titers in nervous tissue, highlight the neurotropism of DTV in this mouse model. Additionally, disease outcome for DTV-infected mice was not affected by sex, as males and females were equally susceptible to disease. This is the first study to comprehensively characterize the clinical disease outcome in a small animal model across a spectrum of POWV/DTV infection doses. Here, we developed a small animal model for DTV pathogenesis that mimics the manifestations of POWV disease in humans. Since it is currently not known whether DTV and POWV differ in their capacity to cause human disease, the animal model detailed in our study could be utilized in future comparative pathogenesis studies, or as a platform for testing the efficacy of vaccines, and anti-virals.

Utilization of RNA in situ Hybridization to Understand the Cellular Localization of Powassan Virus RNA at the Tick-Virus-Host Interface.

Skin is the interface between an attached, feeding tick and a host; consequently, it is the first line of defense against invading pathogenic microorganisms that are delivered to a vertebrate host together with tick saliva. Central to the successful transmission of a tick-borne pathogen are complex interactions between the host immune response and early tick-mediated immunomodulation, all of which initially occur at the skin interface. The focus of this work was to demonstrate the use of RNA in situ hybridization (RNA ISH) as a tool for understanding the cellular localization of viral RNA at the feeding site of Powassan virus (POWV)-infected Ixodes scapularis ticks. Intense positive staining for POWV RNA was frequently detected in dermal foci and occasionally detected in hypodermal foci after 24 h of POWV-infected tick feeding. Additionally, duplex chromogenic RNA ISH staining demonstrated co-localization of POWV RNA with Mus musculus F4/80 RNA, CD11c RNA, vimentin RNA, Krt14 RNA, and CD3ε RNA at the feeding site of POWV-infected ticks. In future studies, RNA ISH can be used to validate transcriptomic analyses conducted at the tick-virus-host cutaneous interface and will provide cellular resolution for specific gene signatures temporally expressed during infected tick feeding. Such a systems biology approach will help create a more refined understanding of the cellular and molecular interactions influencing virus transmission at the cutaneous interface.

Detection of Rickettsiae, Borreliae, and Ehrlichiae in Ticks Collected from Walker County, Texas, 2017-2018.

Cases of tick-borne diseases, including spotted fever rickettsioses, borreliosis, babesiosis, anaplasmosis and ehrlichiosis, in the United States and territories have more than doubled from 2004 to 2016 and account for 77% of all vector-borne disease reports. In an effort to inform control efforts, the presence of tick-borne pathogens and their vectors was assessed in a recreational park in Walker County, Texas. Here we report data from questing ticks collected on three dates from June 2017 to June 2018. The majority of ticks collected were Amblyomma americanum (96.69%) followed by three additional tick species: Dermacentor variabilis (2.59%), Ixodes scapularis (0.52%), and A. maculatum (0.21%). Ticks were pooled and tested for molecular evidence of bacterial and viral pathogens, respectively. All of the 68 pools of A. americanum had molecular evidence of the spotted fever group rickettsia, Rickettsia amblyommatis. Additionally, six (8.82%) of the A. americanum pools contained sequences matching Ehrlichia chaffeensis, the pathogen responsible for human monocytotropic ehrlichiosis, and 11 (16.18%) for E. ewingii. Three of the A. americanum pools demonstrated evidence of Borrelia lonestari. The presence of etiologic agents of known human disease in this study merits the continued surveillance efforts of ticks and their pathogens in areas where they could pose risks to public health.

Ixodes scapularis salivary gland microRNAs are differentially expressed during Powassan virus transmission.

Successful tick feeding is facilitated by an assortment of pharmacologically-active factors in tick saliva that create an immunologically privileged micro-environment in the host's skin. Through a process known as saliva-assisted transmission, bioactive tick salivary factors modulate the host environment, promoting transmission and establishment of a tick-borne pathogen. This phenomenon was previously demonstrated for Powassan virus (POWV), a North American tick-borne flavivirus that is the causative agent of a severe neuroinvasive disease in humans. Here, we sought to characterize the Ixodes scapularis salivary gland microRNAs (miRNAs) expressed during the earliest period of POWV transmission to a mammalian host. POWV-infected and uninfected I. scapularis females were fed on naïve mice for 1, 3, and 6 hours, and Illumina next generation sequencing was used to characterize the salivary gland miRNA expression profiles of POWV-infected versus uninfected ticks. 379 salivary miRNAs were detected, of which 338 are reported here as putative novel I. scapularis miRNAs. 35 salivary gland miRNAs were significantly up-regulated and 17 miRNAs were significantly down-regulated in response to POWV infection. To investigate the potential role of salivary gland miRNAs in POWV replication in-vitro, we transfected miRNA inhibitors into VeroE6 cells to profile temporal POWV replication in mammalian cells. Together, the small RNA sequencing data and the in vitro miRNA inhibition assay suggest that the differentially expressed tick salivary miRNAs could act in regulating POWV replication in host tissues.

Tick⁻Virus⁻Host Interactions at the Cutaneous Interface: The Nidus of Flavivirus Transmission.

Tick-borne viral diseases continue to emerge in the United States, as clearly evident from the increase in Powassan encephalitis virus, Heartland virus, and Bourbon virus infections. Tick-borne flaviviruses (TBFVs) are transmitted to the mammalian host along with the infected tick saliva during blood-feeding. Successful tick feeding is facilitated by a complex repertoire of pharmacologically active salivary proteins/factors in tick saliva. These salivary factors create an immunologically privileged micro-environment in the host's skin that influences virus transmission and pathogenesis. In this review, we will highlight tick determinants of TBFV transmission with a special emphasis on tick⁻virus⁻host interactions at the cutaneous interface.

Transcriptional Immunoprofiling at the Tick-Virus-Host Interface during Early Stages of Tick-Borne Encephalitis Virus Transmission.

Emerging and re-emerging diseases transmitted by blood feeding arthropods are significant global public health problems. Ticks transmit the greatest variety of pathogenic microorganisms of any blood feeding arthropod. Infectious agents transmitted by ticks are delivered to the vertebrate host together with saliva at the bite site. Tick salivary glands produce complex cocktails of bioactive molecules that facilitate blood feeding and pathogen transmission by modulating host hemostasis, pain/itch responses, wound healing, and both innate and adaptive immunity. In this study, we utilized Illumina Next Generation Sequencing to characterize the transcriptional immunoprofile of cutaneous immune responses to Ixodes ricinus transmitted tick-borne encephalitis virus (TBEV). A comparative immune gene expression analysis of TBEV-infected and uninfected tick feeding sites was performed. Our analysis reveals that ticks create an inflammatory environment at the bite site during the first 3 h of feeding, and significant differences in host responses were observed between TBEV-infected and uninfected tick feeding. Gene-expression analysis reveals modulation of inflammatory genes after 1 and 3 h of TBEV-infected tick feeding. Transcriptional levels of genes specific to chemokines and cytokines indicated a neutrophil-dominated immune response. Immunohistochemistry of the tick feeding site revealed that mononuclear phagocytes and fibroblasts are the primary target cells for TBEV infection and did not detect TBEV antigens in neutrophils. Together, the transcriptional and immunohistochemistry results suggest that early cutaneous host responses to TBEV-infected tick feeding are more inflammatory than expected and highlight the importance of inflammatory chemokine and cytokine pathways in tick-borne flavivirus transmission.

An Overview of Animal Models for Arthropod-Borne Viruses.

Arthropod-borne viruses (arboviruses) have continued to emerge in recent years, posing a significant health threat to millions of people worldwide. The majority of arboviruses that are pathogenic to humans are transmitted by mosquitoes and ticks, but other types of arthropod vectors can also be involved in the transmission of these viruses. To alleviate the health burdens associated with arbovirus infections, it is necessary to focus today's research on disease control and therapeutic strategies. Animal models for arboviruses are valuable experimental tools that can shed light on the pathophysiology of infection and will enable the evaluation of future treatments and vaccine candidates. Ideally an animal model will closely mimic the disease manifestations observed in humans. In this review, we outline the currently available animal models for several viruses vectored by mosquitoes, ticks, and midges, for which there are no standardly available vaccines or therapeutics.

Powassan Virus: An Emerging Arbovirus of Public Health Concern in North America.

Powassan virus (POWV, Flaviviridae) is the only North American member of the tick-borne encephalitis serogroup of flaviviruses. It is transmitted to small- and medium-sized mammals by Ixodes scapularis, Ixodes cookei, and several other Ixodes tick species. Humans become infected with POWV during spillover transmission from the natural transmission cycles. In humans, POWV is the causative agent of a severe neuroinvasive illness with 50% of survivors displaying long-term neurological sequelae. POWV was recognized as a human pathogen in 1958 when a young boy died of severe encephalitis in Powassan, Ontario, and POWV was isolated from the brain autopsy of this case. Two distinct genetic lineages of POWV are now recognized: POWV (lineage I) and deer tick virus (lineage II). Since the index case in 1958, over 100 human cases of POWV have been reported, with an apparent rise in disease incidence in the past 16 years. This recent increase in cases may represent a true emergence of POWV in regions where the tick vector species are prevalent, or it could represent an increase in POWV surveillance and diagnosis. In the past 5 years, both basic and applied research for POWV disease has intensified, including phylogenetic studies, field surveillance, case studies, and animal model development. This review provides an overview of POWV, including the epidemiology, transmission, clinical disease, and diagnosis of POWV infection. Recent research developments and future priorities with regard to the disease are emphasized.

Spinal Cord Ventral Horns and Lymphoid Organ Involvement in Powassan Virus Infection in a Mouse Model.

Powassan virus (POWV) belongs to the family Flaviviridae and is a member of the tick-borne encephalitis serogroup. Transmission of POWV from infected ticks to humans has been documented in the USA, Canada, and Russia, causing fatal encephalitis in 10% of human cases and significant neurological sequelae in survivors. We used C57BL/6 mice to investigate POWV infection and pathogenesis. After footpad inoculation, infected animals exhibited rapid disease progression and 100% mortality. Immunohistochemistry and immunofluorescence revealed a very strong neuronal tropism of POWV infection. The central nervous system infection appeared as a meningoencephalitis with perivascular mononuclear infiltration and microglial activation in the brain, and a poliomyelitis-like syndrome with high level of POWV antigen at the ventral horn of the spinal cord. Pathological studies also revealed substantial infection of splenic macrophages by POWV, which suggests that the spleen plays a more important role in pathogenesis than previously realized. This report provides a detailed description of the neuroanatomical distribution of the lesions produced by POWV infection in C57BL/6 mice.

Immune Cell Targets of Infection at the Tick-Skin Interface during Powassan Virus Transmission.

Powassan virus (POWV) is a tick-borne flavivirus that can result in a severe neuroinvasive disease with 50% of survivors displaying long-term neurological sequelae. Human POWV cases have been documented in Canada, the United States, and Russia. Although the number of reported POWV human cases has increased in the past fifteen years, POWV remains one of the less studied human pathogenic flaviviruses. Ixodes ticks are the vectors for POWV, and the virus is transmitted to a host's skin very early during the tick feeding process. Central to the successful transmission of a tick-borne pathogen are complex interactions between the host immune response and early tick-mediated immunomodulation, all of which initially occur at the skin interface. In our prior work, we examined the cutaneous immune gene expression during the early stages of POWV-infected Ixodes scapularis feeding. The present study serves to further investigate the skin interface by identifying early cell targets of infection at the POWV-infected tick feeding site. An in vivo infection model consisting of POWV-infected ticks feeding on mice for short durations was used in this study. Skin biopsies from the tick feeding sites were harvested at various early time points, enabling us to examine the skin histopathology and detect POWV viral antigen in immune cells present at the tick feeding site. The histopathology from the present study demonstrates that neutrophil and mononuclear cell infiltrates are recruited earlier to the feeding site of a POWV-infected tick versus an uninfected tick. This is the first report demonstrating that macrophages and fibroblasts contain POWV antigens, which suggests that they are early cellular targets of infection at the tick feeding site. These data provide key insights towards defining the complex interactions between the host immune response and early tick-mediated immunomodulation.

Tick Saliva Enhances Powassan Virus Transmission to the Host, Influencing Its Dissemination and the Course of Disease.

UNLABELLED: Powassan virus (POWV) is an encephalitic tick-borne flavivirus which can result in serious neuroinvasive disease with up to a 10% case fatality rate. The study objective was to determine whether the salivary gland extract (SGE) from Ixodes scapularis ticks facilitates the transmission and dissemination of POWV in a process known as saliva-activated transmission. Groups of BALB/c mice were footpad inoculated with either a high dose of POWV with and without SGE or a low dose of POWV with and without SGE. Mice from each group were sacrificed daily. Organ viral loads and gene expression profiles were evaluated by quantitative real-time PCR. Both groups of mice infected with high-dose POWV showed severe neurological signs of disease preceding death. The presence of SGE did not affect POWV transmission or disease outcome for mice infected with the high dose of POWV. Neuroinvasion, paralysis, and death occurred for all mice infected with the low dose of POWV plus SGE; however, for mice infected with the low dose of POWV in the absence of SGE, there were no clinical signs of infection and no mice succumbed to disease. Although this group displayed low-level viremias, all mice were completely healthy, and it was the only group in which POWV was cleared from the lymph nodes. We conclude that saliva-activated transmission occurs in mice infected with a low dose of POWV. Our study is the first to demonstrate virus dose-dependent saliva-activated transmission, warranting further investigation of the specific salivary factors responsible for enhancing POWV transmission. IMPORTANCE: Powassan virus (POWV) is a tick-borne flavivirus that continues to emerge in the United States, as is evident by the surge in number and expanding geographic range of confirmed cases in the past decade. This neuroinvasive virus is transmitted to humans by infected tick bites. Successful tick feeding is facilitated by a collection of pharmacologically active factors in tick saliva. In a process known as saliva-activated transmission, tick bioactive salivary molecules are thought to modulate the host environment, making it more favorable for the transmission and establishment of a pathogen. This phenomenon has been demonstrated for several tick-borne pathogens; however, a systematic investigation of the role of tick saliva on dissemination and pathogenesis of a tick-borne viral disease has never been attempted before. This study will fill that gap by systematically examining whether the presence of tick saliva contributes to the transmission and dissemination of POWV in mice.

A novel live-attenuated vaccine candidate for mayaro Fever.

Mayaro virus (MAYV) is an emerging, mosquito-borne alphavirus that causes a dengue-like illness in many regions of South America, and which has the potential to urbanize. Because no specific treatment or vaccine is available for MAYV infection, we capitalized on an IRES-based approach to develop a live-attenuated MAYV vaccine candidate. Testing in infant, immunocompetent as well as interferon receptor-deficient mice demonstrated a high degree of attenuation, strong induction of neutralizing antibodies, and efficacy against lethal challenge. This vaccine strain was also unable to infect mosquito cells, a major safety feature for a live vaccine derived from a mosquito-borne virus. Further preclinical development of this vaccine candidate is warranted to protect against this important emerging disease.