Meghan E. Hermance

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

ABSTRACT 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.

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.

Tick-Borne Viruses and Biological Processes at the Tick-Host-Virus Interface

Ticks are efficient vectors of arboviruses, although less than 10% of tick species are known to be virus vectors. Most tick-borne viruses (TBV) are RNA viruses some of which cause serious diseases in humans and animals world-wide. Several TBV impacting human or domesticated animal health have been found to emerge or re-emerge recently. In order to survive in nature, TBV must infect and replicate in both vertebrate and tick cells, representing very different physiological environments. Information on molecular mechanisms that allow TBV to switch between infecting and replicating in tick and vertebrate cells is scarce. In general, ticks succeed in completing their blood meal thanks to a plethora of biologically active molecules in their saliva that counteract and modulate different arms of the host defense responses (haemostasis, inflammation, innate and acquired immunity, and wound healing). The transmission of TBV occurs primarily during tick feeding and is a complex process, known to be promoted by tick saliva constituents. However, the underlying molecular mechanisms of TBV transmission are poorly understood. Immunomodulatory properties of tick saliva helping overcome the first line of defense to injury and early interactions at the tick-host skin interface appear to be essential in successful TBV transmission and infection of susceptible vertebrate hosts. The local host skin site of tick attachment, modulated by tick saliva, is an important focus of virus replication. Immunomodulation of the tick attachment site also promotes co-feeding transmission of viruses from infected to non-infected ticks in the absence of host viraemia (non-viraemic transmission). Future research should be aimed at identification of the key tick salivary molecules promoting virus transmission, and a molecular description of tick-host-virus interactions and of tick-mediated skin immunomodulation. Such insights will enable the rationale design of anti-tick vaccines that protect against disease caused by tick-borne viruses.

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.

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.

Concurrent micro-RNA mediated silencing of tick-borne flavivirus replication in tick vector and in the brain of vertebrate host.

Tick-borne viruses include medically important zoonotic pathogens that can cause life-threatening diseases. Unlike mosquito-borne viruses, whose impact can be restrained via mosquito population control programs, for tick-borne viruses only vaccination remains the reliable means of disease prevention. For live vaccine viruses a concern exists, that spillovers from viremic vaccinees could result in introduction of genetically modified viruses into sustainable tick-vertebrate host transmission cycle in nature. To restrict tick-borne flavivirus (Langat virus, LGTV) vector tropism, we inserted target sequences for tick-specific microRNAs (mir-1, mir-275 and mir-279) individually or in combination into several distant regions of LGTV genome. This caused selective attenuation of viral replication in tick-derived cells. LGTV expressing combinations of target sequences for tick- and vertebrate CNS-specific miRNAs were developed. The resulting viruses replicated efficiently and remained stable in simian Vero cells, which do not express these miRNAs, however were severely restricted to replicate in tick-derived cells. In addition, simultaneous dual miRNA targeting led to silencing of virus replication in live Ixodes ricinus ticks and abolished virus neurotropism in highly permissive newborn mice. The concurrent restriction of adverse replication events in vertebrate and invertebrate hosts will, therefore, ensure the environmental safety of live tick-borne virus vaccine candidates.

Detection of Rickettsia amblyommii in ticks collected from Missouri, USA.

Dear Editor, In September 2012, we collected ticks from Missouri with the goal of isolating the novel phlebovirus, Heartland virus (HRTV). HRTV was described in two farmers from northwestern Missouri who presented with thrombocytopenia and severe febrile illness.1 These patients were both bitten by ticks 5–7 days before the onset of their clinical symptoms. Our hypothesis was that we would isolate HRTV from ticks collected in Missouri by inoculation of cell culture and/or by detection of viral RNA on polymerase chain reaction (PCR) assay. Furthermore, we used this field surveillance study as an opportunity to screen for other potential viral and bacterial pathogens in the tick samples we collected. Information from the published literature1 was used to identify three geographically relevant collection sites across the central and western region of the state (Supplementary Figure S1A). One thousand two hundred and sixty-nine total ticks were collected from the three locations. Of these, 1191 (93.9%) were Dermacentor albipictus, 74 (5.8%) were Amblyomma americanum and four (0.3%) were Ixodes scapularis (Supplementary Figure S1B). Two nymphs were collected at location 2, but all other ticks collected during this study were larvae. The speciated ticks were pooled into groups of twenty and screened for tick-borne pathogens (Supplementary Methods and Supplementary Table S1). Using the primers specific for HRTV,1 Powassan virus2 and deer tick virus,3 we were unable to generate any positive PCR amplicons in the viral PCR screening. At location 2, one larval pool and one nymphal pool of ticks generated positive PCR amplicons when screened with the Rickettsia-specific primers for the outer membrane protein A (ompA) and citrate synthase (gltA) genes.4 Sequence analysis demonstrated that the two Rickettisa-positive samples aligned with the ompA and gltA genes of Candidatus Rickettsia amblyommii [GenBank: 378930552]. ompA gene sequences for R. amblyommii, R. raoultii, R. slovaca and R. rickettsii were obtained from GenBank. These sequences were trimmed and then underwent ClustalW alignment in the MegAlign program. Specifically, ompA-positive samples from both the larval and nymphal pools shared 100% sequence identity across a 431 bp segment of the ompA gene of Candidatus Rickettsia amblyommii [GenBank: 378930552] (Figure 1A). Additional sequence analysis demonstrated that there was 100% sequence identity between both the larval and nymphal gltA-positive samples and Candidatus Rickettsia amblyommii [GenBank: 378930552] across a 628 bp segment of the gltA gene (Supplementary Figure S2). Furthermore, the presence of Rickettsia in a larval pool of ticks collected at location 2 indicates the occurrence of transovarial transmission of R. amblyommii. Figure 1 Molecular detection of R. amblyommii. (A) Multiple sequence alignment of the outer membrance protein A (ompA) gene. Sequence ruler applies to R. amblyommii omp A sequence. (B) R. amblyommii polyclonal antibody-stained tick homogenate. Tick homogenates ... To further confirm our molecular identification of R. amblyommii, we screened the tick samples with Rickettsia-specific primers for the outer membrane protein B (ompB) gene5 and for the 17 kDa gene.6 The tick samples aligned perfectly with the Candidatus Rickettsia amblyommii [GenBank: 378930552] ompB gene sequence (Supplementary Figure S3). Our R. amblyommii-positive samples also completely aligned with the Candidatus Rickettsia amblyommii [GenBank: 378930552] 17 kDa gene sequence (Supplementary Figure S4). No cytopathic effect was detected in any of the cell lines inoculated with the tick homogenates. However, we used an immunofluorescence assay (Supplementary Methods) to confirm the detection of R. amblyommii antigens in the A. americanum tick homogenates (Figure 1B). Tick mitochondrial 16S rRNA sequence analysis confirmed that the R. amblyommii-positive samples were isolated from A. americanum ticks. The 16S rRNA sequence from our Rickettsia-positive tick pools shared 100% sequence identity with A. americanum 16S rRNA (Supplementary Figure S5). This field surveillance study was unable to isolate HRTV in any of the ticks we collected from Missouri. The lack of HRTV found in this study may be the result of our small sample sizes; only 5.8% of the total collected ticks were A. americanum. After our field collection of ticks was completed, another group published their findings of detecting HRTV from A. americanum collected in Missouri during 2012.7 This group conducted tick collections ranging from April to early-August 2012. As our collection did not occur until mid-September, the majority of ticks collected in our study were larvae, as would be expected.8 We did confirm the presence of R. amblyommii in two pooled A. americanum tick homogenates. To date, no definitive role has been defined for R. amblyommii in human pathogenesis, but a recent study has shown that A. americanum ticks parasitizing humans are frequently infected with R. amblyommii.9 Two A. americanum ticks collected in Kansas were found to be concurrently infected with R. rickettsii, which causes Rocky Mountain spotted fever, and with R. amblyommii.10 The co-infection of these A. americanum ticks with R. rickettsii and R. amblyommii raises interesting questions about the epidemiology of spotted fever group rickettsiae and Rocky Mountain spotted fever. A recent study in North Carolina screened ticks for spotted fever group rickettsiae and found that there was a high prevalence of A. americanum ticks infected with R. amblyommii.11 As this tick species is relatively aggressive and readily parasitizes humans, the authors suggested that some cases of rickettsiosis diagnosed as Rocky Mountain spotted fever in North Carolina may instead be caused by R. amblyommii. Because other Rickettsia species, such as R. parkeri, were initially thought to be endosymbionts but were later shown to be pathogenic, it is important to continue evaluating the potential public health threat that R. amblyommii-infected A. americanum ticks pose to the humans they parasitize.

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.

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.