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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.

A better understanding of the mechanisms used by Ebola virus to disable the host immune system and spread the infection are of great importance for development of new therapeutic strategies. We demonstrate that treatment of monocytic cells with Ebola virus shed glycoprotein (GP) promotes their differentiation resulting in increased infection and cell death. The effects were inhibited by blocking Toll-like receptor 4 pathway. In addition, high levels of shed GP were detected in supernatants of cells treated with Ebola vaccines. This study highlights the role of shed GP in Ebola pathogenesis and also in adverse effects associated with Ebola vaccines.

Ebola virus (EBOV) infections are characterized by a pronounced lymphopenia that is highly correlative with fatalities. However, the mechanisms leading to T-cell depletion remain largely unknown. Here, we demonstrate that both viral mRNAs and antigens are detectable in CD4+ T cells despite the absence of productive infection. A protein phosphatase 1 inhibitor, 1E7-03, and siRNA-mediated suppression of viral antigens were used to demonstrate de novo synthesis of viral RNAs and antigens in CD4+ T cells, respectively. Cell-to-cell fusion of permissive Huh7 cells with non-permissive Jurkat T cells impaired productive EBOV infection suggesting the presence of a cellular restriction factor. We determined that viral transcription is partially impaired in the fusion T cells. Lastly, we demonstrate that exposure of T cells to EBOV resulted in autophagy through activation of ER-stress related pathways. These data indicate that exposure of T cells to EBOV results in an abortive infection, which likely contributes to the lymphopenia observed during EBOV infections., Author summary Lymphopenia is a common characteristic of the disease caused by EBOV. We determined that despite the apparent lack of productive infection, EBOV is capable of entering T cells and producing both viral RNAs and proteins. Furthermore, we demonstrate that EBOV causes an abortive infection in T cells due to the presence of a cellular restriction factor. The abortive infection was associated with cell death following ER-stress induced autophagy. Collectively, these findings suggest that abortive infection in T cells is likely to contribute to lymphopenia during Ebola virus disease, which is uniformly linked with the severity of the disease. All EBOV vaccine candidates utilize GP as the sole antigen inducing a protective antibody response and in some clinical trials were shown to induce adverse side effects. The present study suggests that these effects can be associated with GP, which may lead to abortive infection of the vaccine construct in T cells contributing to the inflammatory response to the vaccines.

Oropouche virus (OROV) is a frequent cause of arboviral febrile disease in the Amazon. The present report describes studies done in two patients, one of them; the first OROV human case acquired outside of the Amazon, which have revealed for the first time the presence of OROV in peripheral blood leukocytes. This novel finding raises important issues regarding pathogenesis of human infections and may offer a new tool, for the rapid diagnosis of this neglected infection. J. Med. Virol. 89:1108-1111, 2017. © 2017 Wiley Periodicals, Inc.

Marburg virus (MARV) and Ebola virus (EBOV) belong to the family Filoviridae. MARV causes severe disease in humans with high fatality. We previously isolated a large panel of monoclonal antibodies (mAbs) from B cells of a human survivor with previous naturally acquired MARV infection. Here, we characterized functional properties of these mAbs and identified non-neutralizing mAbs targeting the glycoprotein (GP) 2 portion of the mucin-like domain (MLD) of MARV GP, termed the wing region. One mAb targeting the GP2 wing, MR228, showed therapeutic protection in mice and guinea pigs infected with MARV. The protection was mediated by the Fc fragment functions of MR228. Binding of another GP2 wing-specific non-neutralizing mAb, MR235, to MARV GP increased accessibility of epitopes in the receptor-binding site (RBS) for neutralizing mAbs, resulting in enhanced virus neutralization by these mAbs. These findings highlight an important role for non-neutralizing mAbs during natural human MARV infection.

Viral apoptotic mimicry, which is defined by exposure of phosphatidylserine (PtdSer) into the outer leaflet of budding enveloped viruses, increases viral tropism, infectivity and promotes immune evasion. Here, we report that the calcium (Ca2+)-dependent scramblase, transmembrane protein 16F (TMEM16F), is responsible for the incorporation of PtdSer into virion membranes during Ebola virus infection. Infection of Huh7 cells with Ebola virus resulted in a pronounced increase in plasma membrane-associated PtdSer, which was demonstrated to be dependent on TMEM16F function. Analysis of virions using imaging flow cytometry revealed that short hairpin RNA-mediated down-regulation of TMEM16F function directly reduced virion-associated PtdSer. Taken together, these studies demonstrate that TMEM16F is a central cellular factor in the exposure of PtdSer in the outer leaflet of viral membranes.

The outer leaflet of the viral membrane of Ebola virus (EBOV) virions is enriched with phosphatidylserine (PtdSer), which is thought to play a central role in viral tropism, entry, and virus-associated immune evasion. We investigated the effects of inhibiting synthesis and/or export of PtdSer to the cell surface of infected cells on viral infectivity. Knockdown of both PtdSer synthase enzymes, PTDSS1 and PTDSS2, effectively decreased viral production. Decreased PtdSer expression resulted in an accumulation of virions at the plasma membrane and adjacent of intracellular organelles, suggesting that virion budding is impaired. The addition of inhibitors that block normal cellular trafficking of PtdSer to the plasma membrane resulted in a similar accumulation of virions and reduced viral replication. These findings demonstrate that plasma membrane-associated PtdSer is required for efficient EBOV budding, increasing EBOV infectivity, and could constitute a potential therapeutic target for the development of future countermeasures against EBOV.

Ebola virus (EBOV) is a negative-strand RNA virus with significant public health importance. Currently, no therapeutics are available for Ebola, which imposes an urgent need for a better understanding of EBOV biology. Here we dissected the virus-host interplay between EBOV and host RNA-binding proteins. We identified novel EBOV host factors, including Staufen1, which interacts with multiple viral factors and is required for efficient viral RNA synthesis., Ebola virus (EBOV) genome and mRNAs contain long, structured regions that could hijack host RNA-binding proteins to facilitate infection. We performed RNA affinity chromatography coupled with mass spectrometry to identify host proteins that bind to EBOV RNAs and identified four high-confidence proviral host factors, including Staufen1 (STAU1), which specifically binds both 3′ and 5′ extracistronic regions of the EBOV genome. We confirmed that EBOV infection rate and production of infectious particles were significantly reduced in STAU1-depleted cells. STAU1 was recruited to sites of EBOV RNA synthesis upon infection and enhanced viral RNA synthesis. Furthermore, STAU1 interacts with EBOV nucleoprotein (NP), virion protein 30 (VP30), and VP35; the latter two bridge the viral polymerase to the NP-coated genome, forming the viral ribonucleoprotein (RNP) complex. Our data indicate that STAU1 plays a critical role in EBOV replication by coordinating interactions between the viral genome and RNA synthesis machinery.

Summary: Some monoclonal antibodies (mAbs) recovered from survivors of filovirus infections can protect against infection. It is currently unknown whether natural infection also induces some antibodies with the capacity for antibody-dependent enhancement (ADE). A panel of mAbs obtained from human survivors of filovirus infection caused by Ebola, Bundibugyo, or Marburg viruses was evaluated for their ability to facilitate ADE. ADE was observed readily with all mAbs examined at sub-neutralizing concentrations, and this effect was not restricted to mAbs with a particular epitope specificity, neutralizing capacity, or subclass. Blocking of specific Fcγ receptors reduced but did not abolish ADE that was associated with high-affinity binding antibodies, suggesting that lower-affinity interactions still cause ADE. Mutations of Fc fragments of an mAb that altered its interaction with Fc receptors rendered the antibody partially protective in vivo at a low dose, suggesting that ADE counteracts antibody-mediated protection and facilitates dissemination of filovirus infections. : In this paper, Kuzmina et al. demonstrate that filovirus antibodies from human survivors present at low concentrations are capable of enhancement of infection, suggesting that low levels of antibodies in humans may facilitate virus spread. The enhancement can be caused by antibodies of various epitope specificities, neutralizing capacities, and subclasses. Keywords: Ebola virus, filovirus, antibody, enhancement of infection, FC receptor, epitope

Recent studies suggest that some monoclonal antibodies (mAbs) specific for ebolavirus glycoprotein (GP) can protect experimental animals against infections. Most mAbs isolated from ebolavirus survivors appeared to target the glycan cap or the stalk region of the viral GP, which is the envelope protein and the only antigen inducing virus-neutralizing antibody response. Some of the mAbs were demonstrated to be protective in vivo. Here, a panel of mAbs from four individual survivors of ebolavirus infection that target the glycan cap or stem region were selected for investigation of the mechanisms of their antiviral effect. Comparative characterization of the inhibiting effects on multiple steps of viral replication was performed, including attachment, post-attachment, entry, binding at low pH, post-cleavage neutralization of virions, viral trafficking to endosomes, cell-to-cell transmission, viral egress, and inhibition when added early at various time points post-infection. In addition, Fc-domain related properties were characterized, including activation and degranulation of NK cells, antibody-dependent cellular phagocytosis and glycan content. The two groups of mAbs (glycan cap versus stem) demonstrated very different profiles of activities suggesting usage of mAbs with different epitope specificity could coordinate inhibition of multiple steps of filovirus infection through Fab- and Fc-mediated mechanisms, and provide a reliable therapeutic approach., Author summary Recent progress in isolation of mAbs from survivors of filovirus infections suggests that the human adaptive immune system is capable of producing strong antibody responses. However, the effects of mAbs with different epitope specificity on individual steps of filovirus infection are still unclear. We evaluated a panel of mAbs obtained from survivors of natural filovirus infections, specific for the glycan cap or stem region of GP, for their effects on the attachment of viral particles to the cell surface, intracellular traffic of viral particles, proteolytic processing of GP, its interaction with the NPC1 receptor, cell-to-cell virus transmission, virus egress from infected cells, activation of natural killer cells and antibody-dependent cellular phagocytosis through Fc-mediated mechanisms. We found that antiviral activity of glycan cap-specific antibodies results from inhibition of attachment, cell-to-cell transmission and inhibition of virion budding. In contrast, the antiviral mechanisms of stem-specific antibodies were found to be inhibition of virus release from endosomal network to the cytoplasm, and also activation of natural killer cells and phagocytosis mediated by monocytes and neutrophils. The data provide new insight into the development of immune protective mechanisms during natural human infection, and have important implications for the treatment of filovirus infections by passively-transferred antibodies and vaccine design.

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.

Ebola virus (EBOV) disease (EVD) results from an exacerbated immunological response that is highlighted by a burst in the production of inflammatory mediators known as a “cytokine storm.” Previous reports have suggested that nonspecific activation of T lymphocytes may play a central role in this phenomenon. T-cell immunoglobulin and mucin domain-containing protein 1 (Tim-1) has recently been shown to interact with virion-associated phosphatidylserine to promote infection. Here, we demonstrate the central role of Tim-1 in EBOV pathogenesis, as Tim-1−/− mice exhibited increased survival rates and reduced disease severity; surprisingly, only a limited decrease in viremia was detected. Tim-1−/− mice exhibited a modified inflammatory response as evidenced by changes in serum cytokines and activation of T helper subsets. A series of in vitro assays based on the Tim-1 expression profile on T cells demonstrated that despite the apparent absence of detectable viral replication in T lymphocytes, EBOV directly binds to isolated T lymphocytes in a phosphatidylserine–Tim-1-dependent manner. Exposure to EBOV resulted in the rapid development of a CD4Hi CD3Low population, non-antigen-specific activation, and cytokine production. Transcriptome and Western blot analysis of EBOV-stimulated CD4+ T cells confirmed the induction of the Tim-1 signaling pathway. Furthermore, comparative analysis of transcriptome data and cytokine/chemokine analysis of supernatants highlight the similarities associated with EBOV-stimulated T cells and the onset of a cytokine storm. Flow cytometry revealed virtually exclusive binding and activation of central memory CD4+ T cells. These findings provide evidence for the role of Tim-1 in the induction of a cytokine storm phenomenon and the pathogenesis of EVD., IMPORTANCE Ebola virus infection is characterized by a massive release of inflammatory mediators, which has come to be known as a cytokine storm. The severity of the cytokine storm is consistently linked with fatal disease outcome. Previous findings have demonstrated that specific T-cell subsets are key contributors to the onset of a cytokine storm. In this study, we investigated the role of Tim-1, a T-cell-receptor-independent trigger of T-cell activation. We first demonstrated that Tim-1-knockout (KO) mice survive lethal Ebola virus challenge. We then used a series of in vitro assays to demonstrate that Ebola virus directly binds primary T cells in a Tim-1–phosphatidylserine-dependent manner. We noted that binding induces a cytokine storm-like phenomenon and that blocking Tim-1–phosphatidylserine interactions reduces viral binding, T-cell activation, and cytokine production. These findings highlight a previously unknown role of Tim-1 in the development of a cytokine storm and “immune paralysis.”

Fatal outcomes of Ebola virus (EBOV) infections are typically preceded by a ‘sepsis-like’ syndrome and lymphopenia despite T cells being resistant to Ebola infection. The mechanisms that lead to T lymphocytes death remain largely unknown; however, the degree of lymphopenia is highly correlative with fatalities. Here we investigated whether the addition of EBOV or its envelope glycoprotein (GP) to isolated primary human CD4+ T cells induced cell death. We observed a significant decrease in cell viability in a GP-dependent manner, which is suggestive of a direct role of GP in T cell death. Using immunoprecipitation assays and flow cytometry, we demonstrate that EBOV directly binds to CD4+ T cells through interaction of GP with TLR4. Transcriptome analysis revealed that the addition of EBOV to CD4+ T cells results in the significant upregulation of pathways associated with interferon signaling, pattern recognition receptors and intracellular activation of NFκB signaling pathway. Both transcriptome analysis and specific inhibitors allowed identification of apoptosis and necrosis as mechanisms associated with the observed T cell death following exposure to EBOV. The addition of the TLR4 inhibitor CLI-095 significantly reduced CD4+ T cell death induced by GP. EBOV stimulation of primary CD4+ T cells resulted in a significant increase in secreted TNFα; inhibition of TNFα-mediated signaling events significantly reduced T cell death while inhibitors of both necrosis and apoptosis similarly reduced EBOV-induced T cell death. Lastly, we show that stimulation with EBOV or GP augments monocyte maturation as determined by an overall increase in expression levels of markers of differentiation. Subsequently, the increased rates of cellular differentiation resulted in higher rates of infection further contributing to T cell death. These results demonstrate that GP directly subverts the host’s immune response by increasing the susceptibility of monocytes to EBOV infection and triggering lymphopenia through direct and indirect mechanisms., Author summary The latest outbreak of Ebola virus (EBOV) in West Africa resulted in more than 28,000 human infections including more than 11,000 deaths thus highlighting the necessity for the development of countermeasures. Monocytes and dendritic cells are among the primary targets of EBOV infection; infection of these critical antigen presenting cells contributes to the immune deficiency observed in Ebola virus disease (EVD). In contrast, lymphocytes are resistant to EBOV infection; however, in fatal EVD, pronounced lymphopenia is uniformly observed. Here we report that T lymphocyte cell death in the absence of detectable infection was observed in an EBOV glycoprotein (GP)-dependent manner. Using transcriptome analysis of EBOV-stimulated CD4+ T cells we show upregulation of both toll-like receptor 4 (TLR4) and cell death associated pathways. Furthermore, we demonstrate that EBOV increases susceptibility of monocytes to infection by promoting cellular differentiation. Both EBOV-induced monocyte differentiation and cell death of T lymphocytes result from a direct interaction between GP and TLR4. Blocking of TLR4 signaling significantly reduced both EBOV-induced T cell death and infection of monocytes. These data contribute to understanding of the ‘immune paralysis’ during EBOV infections and provide evidence for the development of targeted therapies for the treatment of EVD.

Oropouche virus (OROV), of the family Bunyaviridae, is the second most frequent arbovirus causing febrile disease in Brazil. In spite of this, little is known about pathogenesis of OROV infection. This report describes an experimental model of OROV in golden hamster (Mesocricetus auratus). Following subcutaneous inoculation of OROV, over 50% of the animals developed disease characterized by lethargy, ruffled fur, shivering, paralysis, and approximately one third died. Animals were sacrificed on days 1, 3, 5, 8 and 11 post-inoculation to collect tissue samples from brain, heart, liver, lung, spleen, muscle and blood for virus titration, histology and OROV immunohistochemistry. OROV was detected in high titers in blood, liver and brain, but not in the other organs. Histopathology revealed meningoencephalitis and hepatitis, with abundant OROV antigen detected in liver and brain. Diffuse galectin-3 immunostaining in brain and liver supports microglial and Kupfer cells activation. This is the first description of an experimental model for OROV infection and should be helpful to study pathogenesis and possibly to test antiviral interventions such as drugs and vaccine candidates.

Oropouche virus (OROV) is an important cause of arboviral illness in Brazil and other Latin American countries, with most cases clinically manifested as acute febrile illness referred to as Oropouche fever, including myalgia, headache, arthralgia and malaise. However, OROV can also affect the central nervous system (CNS) with clinical neurological implications. Little is known regarding OROV pathogenesis, especially how OROV gains access to the CNS. In the present study, neonatal BALB/c mice were inoculated with OROV by the subcutaneous route and the progression of OROV spread into the CNS was evaluated. Immunohistochemistry revealed that OROV infection advances from posterior parts of the brain, including the periaqueductal gray, toward the forebrain. In the early phases of the infection OROV gains access to neural routes, reaching the spinal cord and ascending to the brain through brainstem regions, with little inflammation. Later, as infection progresses, OROV crosses the blood-brain barrier, resulting in more intense spread into the brain parenchyma, with more severe manifestations of encephalitis.

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.

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.

Oropouche (OROV) is a single-stranded RNA arbovirus of the family Bunyaviridae, genus Orthobunyavirus, which has caused over half a million cases of febrile illness in Brazil in the past 30 years. OROV fever has been registered almost exclusively in the Amazon region, but global warming, deforestation and redistribution of vectors and animal reservoirs increases the risk of Oropouche virus emergence in other areas. OROV causes a cytolytical infection in cultured cells with characteristic cytopathic effect 48 h post-infection. We have studied the mechanisms of apoptosis induced by OROV in HeLa cells and found that OROV causes DNA fragmentation detectable by gel electrophoresis and by flow cytometric analysis of the Sub-G1 population at 36 h post-infection. Mitochondrial release of cytochrome C and activation of caspases 9 and 3 were also detected by western blot analysis. Lack of apoptosis induced by UV-inactivated OROV reveals that virus-receptor binding is not sufficient to induce cell death. Results obtained in cells treated with chloroquine and cycloheximide indicated that viral uncoating and replication are required for apoptosis induction by OROV. Furthermore, treatment of the cells with pan-caspase inhibitor prevented OROV-induced apoptosis without affecting virus progeny production. The results show that OROV infection in vitro causes apoptosis by an intracellular pathway involving mitochondria, and activated by a mechanism dependent on viral replication and protein synthesis.

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.

Oropouche virus, of the family Bunyaviridae, genus Orthobunyavirus, serogroup Simbu, is an important causative agent of arboviral febrile illness in Brazil. An estimated 500,000 cases of Oropouche fever have occurred in Brazil in the last 30 years, with recorded cases also in Panama, Peru, Suriname and Trinidad. We have developed an experimental model of Oropouche virus infection in neonatal BALB/c mouse by subcutaneous inoculation. The vast majority of infected animals developed disease on the 5th day post infection, characterized mainly by lethargy and paralysis, progressing to death within 10 days. Viral replication was documented in brain cells by in situ hybridization, immunohistochemistry and virus titration. Multi-step immunohistochemistry indicated neurons as the main target cells of OROV infection. Histopathology revealed glial reaction and astrocyte activation in the brain and spinal cord, with neuronal apoptosis. Spleen hyperplasia and mild meningitis were also found, without viable virus detected in liver and spleen. This is the first report of an experimental mouse model of OROV infection, with severe involvement of the central nervous system, and should become useful in pathogenesis studies, as well as in preclinical testing of therapeutic interventions for this emerging pathogen.