Nipah virus (NiV) causes a highly lethal disease in humans who present with acute respiratory or neurological signs. No vaccines against NiV have been approved to date. Here, we report on the clinical impact of a novel NiV-derived nonspreading replicon particle lacking the fusion (F) protein gene (NiVΔF) as a vaccine in three small animal models of disease. A broad antibody response was detected that included immunoglobulin G (IgG) and IgA subtypes with demonstrable Fc-mediated effector function targeting multiple viral antigens. Single-dose intranasal vaccination up to 3 days before challenge prevented clinical signs and reduced virus levels in hamsters and immunocompromised mice; decreases were seen in tissues and mucosal secretions, critically decreasing potential for virus transmission. This virus replicon particle system provides a vital tool to the field and demonstrates utility as a highly efficacious and safe vaccine candidate that can be administered parenterally or mucosally to protect against lethal Nipah disease.

We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we reveal the role of N-myristoylation during vaccinia virus (VACV) infection in human host cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors. Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we examine the importance of N-myristoylation during vaccinia virus (VACV) infection in primate cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors.

The different families of viruses can be distinguished morphologically by transmission electron microscopy (EM) and this allows for diagnosis to the level of the family. My career with CDC started in 1983 and I have worked with every family of virus that causes human disease and would like to share my experiences.

The western black-legged tick (Ixodes pacificus) is the most frequently identified human-biting tick species in the western United States and the principal vector of at least three recognized bacterial pathogens of humans. A potentially pathogenic Rickettsia species, first described in 1978 and recently characterized as a novel transitional group agent designated as Rickettsia tillamookensis, also exists among populations of I. pacificus, although the distribution and frequency of this agent are poorly known. We evaluated DNA extracts from 348 host-seeking I. pacificus nymphs collected from 9 locations in five California counties, and from 916 I. pacificus adults collected from 24 locations in 13 counties, by using a real-time PCR designed specifically to detect DNA of R. tillamookensis. DNA of R. tillamookensis was detected in 10 (2.9%) nymphs (95% CI: 1.6-5.2%) and 17 (1.9%) adults (95% CI: 1.2-3.0%) from 11 counties of northern California. Although site-specific infection rates varied greatly, frequencies of infection remained consistently low when aggregated by stage, sex, habitat type, or geographical region. Four novel isolates of R. tillamookensis were cultivated in Vero E6 cells from individual adult ticks collected from Alameda, Nevada, and Yolo counties. Four historical isolates, serotyped previously as 'Tillamook-like' strains over 40 yr ago, were revived from long-term storage in liquid nitrogen and confirmed subsequently by molecular methods as isolates of R. tillamookensis. The potential public health impact of R. tillamookensis requires further investigation.

The identification of viral particles within a tissue specimen requires specific knowledge of viral ultrastructure and replication, as well as a thorough familiarity with normal subcellular organelles. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored how challenging the task of identifying coronavirus by electron microscopy (EM) can be. Numerous articles have been published mischaracterizing common subcellular structures, including clathrin- or coatomer- coated vesicles, multivesicular bodies, and rough endoplasmic reticulum, as coronavirus particles in SARS-CoV-2 positive patient tissue specimens. To counter these misinterpretations, we describe the morphological features of coronaviruses that should be used to differentiate coronavirus particles from subcellular structures. Further, as many of the misidentifications of coronavirus particles have stemmed from attempts to attribute tissue damage to direct infection by SARS-CoV-2, we review articles describing ultrastructural changes observed in specimens from SARS-CoV-2-infected individuals that do not necessarily provide EM evidence of direct viral infection. Ultrastructural changes have been observed in respiratory, cardiac, kidney, and intestinal tissues, highlighting the widespread effects that SARS-CoV-2 infection may have on the body, whether through direct viral infection or mediated by SARS-CoV-2 infection-induced inflammatory and immune processes.

Severe coronavirus disease in neonates is rare. We analyzed clinical, laboratory, and autopsy findings from a neonate in the United States who was delivered at 25 weeks of gestation and died 4 days after birth; the mother had asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and preeclampsia. We observed severe diffuse alveolar damage and localized SARS-CoV-2 by immunohistochemistry, in situ hybridization, and electron microscopy of the lungs of the neonate. We localized SARS-CoV-2 RNA in neonatal heart and liver vascular endothelium by using in situ hybridization and detected SARS-CoV-2 RNA in neonatal and placental tissues by using reverse transcription PCR. Subgenomic reverse transcription PCR suggested viral replication in lung/airway, heart, and liver. These findings indicate that in utero SARS-CoV-2 transmission contributed to this neonatal death.

Individuals with metabolic dysregulation of cellular glycosylation often experience severe influenza disease, with a poor immune response to the virus and low vaccine efficacy. Here, we investigate the consequences of aberrant cellular glycosylation for the glycome and the biology of influenza virus. We transiently induced aberrant N-linked glycosylation in cultured cells with an oligosaccharyltransferase inhibitor, NGI-1. Cells treated with NGI-1 produced morphologically unaltered viable influenza virus with sequence-neutral glycosylation changes (primarily reduced site occupancy) in the hemagglutinin and neuraminidase proteins. Hemagglutinin with reduced glycan occupancy required a higher concentration of surfactant protein D (an important innate immunity respiratory tract collectin) for inhibition compared to that with normal glycan occupancy. Immunization of mice with NGI-1-treated virus significantly reduced antihemagglutinin and antineuraminidase titers of total serum antibody and reduced hemagglutinin protective antibody responses. Our data suggest that aberrant cellular glycosylation may increase the risk of severe influenza as a result of the increased ability of glycome-modified influenza viruses to evade the immune response. IMPORTANCE People with disorders such as cancer, autoimmune disease, diabetes, or obesity often have metabolic dysregulation of cellular glycosylation and also have more severe influenza disease, a reduced immune response to the virus, and reduced vaccine efficacy. Since influenza viruses that infect such people do not show consistent genomic variations, it is generally assumed that the altered biology is mainly related to host factors. However, since host cells are responsible for glycosylation of influenza virus hemagglutinin and neuraminidase, and glycosylation is important for interactions of these proteins with the immune system, the viruses may have functional differences that are not reflected by their genomic sequence. Here, we show that imbalanced cellular glycosylation can modify the viral glycome without genomic changes, leading to reduced innate and adaptive host immune responses to infection. Our findings link metabolic dysregulation of host glycosylation to increased risk of severe influenza and reduced influenza virus vaccine efficacy.

Smallpox, caused by the solely human pathogen Variola virus (VARV), was declared eradicated in 1980. While known VARV stocks are secure, smallpox remains a bioterrorist threat agent. Recent U.S. Food and Drug Administration approval of the first smallpox anti-viral (tecovirimat) therapeutic was a successful step forward in smallpox preparedness; however, orthopoxviruses can become resistant to treatment, suggesting a multi-therapeutic approach is necessary. Animal models are required for testing medical countermeasures (MCMs) and ideally MCMs are tested directly against the pathogen of interest. Since VARV only infects humans, a representative animal model for testing therapeutics directly against VARV remains a challenge. Here we show that three different humanized mice strains are highly susceptible to VARV infection, establishing the first small animal model using VARV. In comparison, the non-humanized, immunosuppressed background mouse was not susceptible to systemic VARV infection. Following an intranasal VARV challenge that mimics the natural route for human smallpox transmission, the virus spread systemically within the humanized mouse before mortality (~ 13 days post infection), similar to the time from exposure to symptom onset for ordinary human smallpox. Our identification of a permissive/representative VARV animal model can facilitate testing of MCMs in a manner consistent with their intended use.

BACKGROUND: Lassa fever is a zoonotic, acute viral illness first identified in Nigeria in 1969. An estimate shows that the "at risk" seronegative population (in Sierra Leone, Guinea, and Nigeria) may be as high as 59 million, with an annual incidence of all illnesses of three million, and fatalities up to 67,000, demonstrating the serious impact of the disease on the region and global health. METHODS: Histopathologic evaluation, immunohistochemical assay, and electron microscopic examination were performed on postmortem tissue samples from 12 confirmed Lassa fever cases. RESULTS: Lassa fever virus antigens and viral particles were observed in multiple organ systems and cells, including cells in the mononuclear phagocytic system and other specialized cells where it had not been described previously. CONCLUSIONS: The immunolocalization of Lassa fever virus antigens in fatal cases provides novel insightful information with clinical and pathogenetic implications. The extensive involvement of the mononuclear phagocytic system, including tissue macrophages and endothelial cells suggests participation of inflammatory mediators from this lineage with the resulting vascular dilatation and increasing permeability. Other findings indicate the pathogenesis of LF is multifactorial and additional studies are needed.

Microsporidia are unicellular obligate parasitic fungi which produce heat-resistant spores. | The spores are infective forms containing a coiled polar tubule, which is extruded in the host cell to inject sporoplasm. | Transmission electron microscopy is the gold standard test for identification of microsporidia. Other tests for identification include Gram stain, Chromotrope 2R, quick-hot Gram chromotrope technique, trichrome blue, acid-fast stain, Warthin-Starry stain, modified trichrome stains, calcofluor white, immunohistochemistry, and PCR. | Encephalitozoon intestinalis commonly infects the gastrointestinal tract. However, it can also infect the kidneys and lungs. Microsporidiosis should be considered as a cause of renal failure in an immunocompromised patient.

Efforts to combat the coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have placed a renewed focus on the use of transmission electron microscopy for identifying coronavirus in tissues. In attempts to attribute pathology of COVID-19 patients directly to tissue damage caused by SARS-CoV-2, investigators have inaccurately reported subcellular structures, including coated vesicles, multivesicular bodies, and vesiculating rough endoplasmic reticulum, as coronavirus particles. We describe morphologic features of coronavirus that distinguish it from subcellular structures, including particle size range (60-140 nm), intracellular particle location within membrane-bound vacuoles, and a nucleocapsid appearing in cross section as dense dots (6-12 nm) within the particles. In addition, although the characteristic spikes of coronaviruses may be visible on the virus surface, especially on extracellular particles, they are less evident in thin sections than in negative stain preparations.

This guidance provides clear, concise strategies for identifying coronaviruses by transmission electron microscopy of ultrathin sections of tissues or infected tissue cultures. These include a description of virus morphology as well as cell organelles that can resemble viruses. Biochemical testing and caveats are discussed. Numerous references provide information for documentation and further study.

We are concerned about the erroneous identification of coronavirus directly in tissues by authors using electron microscopy. Several recent articles have been published that purport to have identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly in tissue.1–234 Most describe particles that resemble, but do not have the appearance of, coronaviruses.5–67

An ongoing pandemic of coronavirus disease (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Characterization of the histopathology and cellular localization of SARS-CoV-2 in the tissues of patients with fatal COVID-19 is critical to further understand its pathogenesis and transmission and for public health prevention measures. We report clinicopathologic, immunohistochemical, and electron microscopic findings in tissues from 8 fatal laboratory-confirmed cases of SARS-CoV-2 infection in the United States. All cases except 1 were in residents of long-term care facilities. In these patients, SARS-CoV-2 infected epithelium of the upper and lower airways with diffuse alveolar damage as the predominant pulmonary pathology. SARS-CoV-2 was detectable by immunohistochemistry and electron microscopy in conducting airways, pneumocytes, alveolar macrophages, and a hilar lymph node but was not identified in other extrapulmonary tissues. Respiratory viral co-infections were identified in 3 cases; 3 cases had evidence of bacterial co-infection.

We read with interest the Correspondence by Zsuzsanna Varga and colleagues1 on the possible infection of endothelial cells by SARS-CoV-2 using electron microscopic (EM) images as evidence. However, we believe the EM images in the Correspondence do not show coronavirus particles but instead show cross-sections of the rough endoplasmic reticulum (RER). These spherical structures are surrounded by dark dots, which might have been interpreted as spikes on coronavirus particles but are instead ribosomes. The purported particles are free within the cytoplasm, whereas within a coronavirus-infected cell, accumulations of virus particles would be found in membrane-bound areas in the cisternae of the RER–Golgi area, where the spikes would be located on the inside of the cisternal space.2 In addition, cross-sections through the viral nucleocapsid are not seen in the interior of these structures as would be found with coronavirus particles (figure ).

Microsporidia are a group of obligate intracellular parasites that naturally infect domestic and wild animals. Human microsporidiosis is an increasingly recognized multisystem opportunistic infection. The clinical manifestations are diverse with diarrhea being the most common presenting symptom. We present a 52-year-old woman with a history of amyopathic dermatomyositis complicated by interstitial lung disease managed with mycophenolate mofetil and hydroxychloroquine who presented with a seven-month history of recurrent subcutaneous nodules as well as intermittent diarrhea and chronic sinusitis. A punch biopsy demonstrated superficial and deep lymphocytic and granulomatous dermatitis with focal necrosis. Tissue stains for microorganisms revealed oval 1-3 mum spores within the necrotic areas in multiple tissue stains. Additional studies at the Centers for Disease Control confirmed cutaneous microsporidiosis. This case is one of very few confirmed examples of cutaneous microsporidiosis reported in the literature. This article is protected by copyright. All rights reserved.

The recent reduction of live attenuated influenza vaccine (LAIV) effectiveness in multivalent formulations was particularly associated with the A(H1N1)pdm09 component. In the 2017 the WHO vaccine composition committee changed its recommendations for the A(H1N1)pdm09 component to include an A/Michigan/45/2015-like virus. We evaluated effectiveness and quality of newly developed and previous A(H1N1)pdm09 LAIV reassortants through assessment of their thermal and pH stability, receptor binding specificity and replication fitness in primary human airway epithelial cells of nasal origin (hAECN). Our analysis showed that LAIV expressed hemagglutinin (HA) and neuraminidase (NA) from an A/Michigan/45/2015-like strain A/New York/61/2015 (A/New York/61/2015-CDC-LV16A, NY-LV16A), exhibit higher thermal and pH stability compared to the previous vaccine candidates expressing HA and NA from A/California/07/2009 and A/Bolivia/559/2013 (A17/Cal09 and A17/Bol13). Reassortants A/South Africa/3626/2013-CDC-LV14A (SA-LV14A) and NY-LV16A showed preferential binding to alpha2,6 sialic acid (SA) receptors and replicated at higher titers and more extensively in hAECN compared to A17/Cal09 and A17/Bol13, which had an alpha2,3 SA receptor binding preference. Our data analysis supports selection of A/New York/61/2015-CDC-LV16A for LAIV formulation and the introduction of new assays for LAIV characterization.

Lassa fever is a frequently severe human disease that is endemic to several countries in West Africa. To date, no licensed vaccines are available to prevent Lassa virus (LASV) infection, even though Lassa fever is thought to be an important disease contributing to mortality and both acute and chronic morbidity. We have previously described a vaccine candidate composed of single-cycle LASV replicon particles (VRPs) and a stable cell line for their production. Here, we refine the genetic composition of the VRPs and demonstrate the ability to reproducibly purify them with high yields. Studies in the guinea pig model confirm efficacy of the vaccine candidate, demonstrate that single-cycle replication is necessary for complete protection by the VRP vaccine, and show that post-exposure vaccination can confer protection from lethal outcome.

In 2011, ticks were collected from livestock following an outbreak of Crimean Congo Hemorrhagic fever (CCHF) in Gujarat state, India. CCHF-negative Hyalomma anatolicum tick pools were passaged for virus isolation, and two virus isolates were obtained, designated Karyana virus (KARYV) and Kundal virus (KUNDV) respectively. Traditional RT-PCR identification of known viruses was unsuccessful, but a next-generation sequencing approach identified KARYV and KUNDV as viruses in the Reoviridae family, Orbivirus, and Coltivirus genera, respectively. Viral genomes were de novo assembled, yielding 10 complete segments of KARYV and 12 nearly complete segments of KUNDV. The VP1 gene of KARYV shared a most recent common ancestor with Wad Medani virus (WMV), strain Ar495, and based on nucleotide identity we demonstrate that it is a novel WMV strain. The VP1 segment of KUNDV shares a common ancestor with Colorado tick fever virus, Eyach virus, Tai Forest reovirus and Tarumizu tick virus from the Coltivirus genus. Based on VP1, VP6, VP7, and VP12 nucleotide and amino acid identity, KUNDV is proposed to be a new species of Coltivirus Electron microscopy supported the classification of KARYV and KUNDV as reoviruses and identified replication morphology consistent with other Orbi- and Colti- viruses. The identification of novel tick-borne viruses carried by the CCHF vector is an important step in the characterization of their potential role in human and animal pathogenesis.Importance Ticks, mosquitoes, as well Culicoides, can transmit viruses in the Reoviridae family. With the help of next-generation sequencing (NGS), previously unreported reoviruses such as equine encephalosis virus, Wad Medani virus (WMV), Kammanvanpettai virus (KVPTV) and with this report, KARYV and KUNDV have been discovered and characterized in India. The isolation of KUNDV and KARYV from Hyalomma anatolicum, which is a known vector for zoonotic pathogens, such as Crimean Congo Hemorrhagic Fever virus, Babesia, Theileria and Anaplasma species, identifies arboviruses with the potential to transmit to humans. Characterization of these KUNDV and KARYV isolated from Hyalomma ticks is critical for the development of specific serological and molecular assays that can be used to determine the association of these viruses with disease in humans and livestock.

Ferrets represent an invaluable animal model to study influenza virus pathogenesis and transmission. To further characterize this model, we developed a differentiated primary ferret nasal epithelial cell (FNEC) culture model for investigation of influenza A virus infection and virus-host interactions. This well-differentiated culture consists of various cell types, a mucociliary clearance system, and tight junctions, representing the nasal ciliated pseudostratified respiratory epithelium. Both alpha2,6-linked and alpha2,3-linked sialic acid (SA) receptors, which preferentially bind the HA of human and avian influenza viruses, respectively, were detected on the apical surface of the culture with different cellular tropism. In accordance with distribution of SA receptors, we observed that a pre-2009 seasonal A(H1N1) virus infected both ciliated and non-ciliated cells, whereas a highly pathogenic avian influenza (HPAI) A(H5N1) virus primarily infected non-ciliated cells. Transmission electron microscopy revealed that virions were released from or associated with the apical membranes of ciliated, non-ciliated, and mucin-secretory goblet cells. Upon infection, the HPAI A(H5N1) virus replicated to titers higher than those of the human A(H1N1) virus at 37 degrees C, however, replication of the A(H5N1) virus was significantly attenuated at 33 degrees C. Furthermore, we found that infection with the A(H5N1) virus induced higher expression of immune mediator genes and resulted in more cell damage/loss when compared with the human A(H1N1) virus. This primary differentiated FNEC culture model, recapitulating the structure of the nasal epithelium, provides a useful model to bridge in vivo and in vitro studies of cellular tropism, infectivity, and pathogenesis of influenza viruses during the initial stages of infection.IMPORTANCE: Although ferrets serve as an important model of influenza virus infection, much remains unknown about virus-host interactions in this species at the cellular level. The development of differentiated primary cultures of ferret nasal epithelial cells is an important step toward understanding cellular tropism and the mechanisms of influenza virus infection and replication in the airway milieu of this model. Using lectin staining and microscopy techniques, we characterized sialic acid receptor distribution and the cellular composition of the culture model. We then evaluated the replication of and immune response to human and avian influenza viruses at relevant physiological temperatures. Our findings offer significant insight into this first line defense against influenza virus infection and provide a model for the evaluation of emerging influenza viruses in a well-controlled in vitro environmental setting.

Neutralizing antibodies (NAbs) are traditionally thought to inhibit virus infection by preventing virion entry into target cells. In addition, antibodies can engage Fc receptors (FcRs) on immune cells to activate antiviral responses. We describe a mechanism by which NAbs inhibit chikungunya virus (CHIKV), the most common alphavirus infecting humans, by preventing virus budding from infected human cells and activating IgG-specific Fcgamma receptors. NAbs bind to CHIKV glycoproteins on the infected cell surface and induce glycoprotein coalescence, preventing budding of nascent virions and leaving structurally heterogeneous nucleocapsids arrested in the cytosol. Furthermore, NAbs induce clustering of CHIKV replication spherules at sites of budding blockage. Functionally, these densely packed glycoprotein-NAb complexes on infected cells activate Fcgamma receptors, inducing a strong, antibody-dependent, cell-mediated cytotoxicity response from immune effector cells. Our findings describe a triply functional antiviral pathway for NAbs that might be broadly applicable across virus-host systems, suggesting avenues for therapeutic innovation through antibody design.

Human protothecosis is a rare microalgae infection, and its dissemination typically occurs in immunocompromised individuals, but no specific immune defect has been reported. Here, we describe an 8-year-old daughter of a consanguineous union with abdominal pain and bloody diarrhea for 3 months who was found to have pancolitis with numerous microalgae identified as Prototheca zopfii. In the absence of a known immunodeficiency, exome sequencing was performed, which uncovered a novel recessive frameshift mutation in CARD9 (p.V261fs). This report highlights that CARD9 deficiency should be investigated in patients with unexplained systemic/visceral protothecosis and suggests a new mechanistic insight into anti-Prototheca immunity.

We have previously described a novel taxon of the genus Ehrlichia (type strain WisconsinT), closely related to Ehrlichia muris, that causes human ehrlichiosis among patients with exposures to ticks in the upper midwestern USA. DNA from this bacterium was also detected in Ixodes scapularis and Peromyscus leucopus collected in Minnesota and Wisconsin. To determine the relationship between the E. muris-like agent (EMLA) and other species of the genus Ehrlichia phenotypic, genotypic and epidemiologic comparisons were undertaken, including sequence analysis of eight gene loci (3906 nucleotides) for 39 EMLA DNA samples and the type strain of E. muris AS145T. Three loci were also sequenced from DNA of nine strains of E. muris from mouse spleens from Japan. All sequences from E. muris were distinct from homologous EMLA sequences, but differences between them were less than those observed among other species of the genus Ehrlichia. Phenotypic comparison of EMLA and E. muris revealed similar culture and electron microscopic characteristics, but important differences were noted in their geographic distribution, ecological associations and behavior in mouse models of infection. Based on these comparisons, we propose that type strain WisconsinT represents a novel subspecies, Ehrlichia murissubsp. eauclairensis,subsp. nov. This strain is available through the Centers for Disease Control and Prevention Rickettsial Isolate Reference Collection (CRIRC EMU002T) and through the Collection de Souches de l'Unite des Rickettsies (CSURP2883 T). The subspecies Ehrlichia murissubsp. muris subsp. nov. is automatically created and the type strain AS145T is also available through the same collections (CRIRC EMU001T, CSUR E2T). Included is an emended description of E. muris.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral hemorrhagic disease seen exclusively in humans. Central nervous system (CNS) infection and neurological involvement have also been reported in CCHF. Here we inoculated NSGTM-SGM3 mice engrafted with human hematopoietic CD34+ stem cells with low passage CCHF virus strains isolated from human patients. Humanized mice develop lethal disease characterized by histopathological change in the liver and brain. To date, targets of neuroinfection and neuropathology have not been investigated in CCHF. CNS disease in humanized mice was characterized by gliosis, meningitis and meningoencephalitis, and glial cells were identified as principal targets of infection. Humanized mice represent a novel lethal model for studies of CCHF countermeasures, and CCHF-associated CNS disease. Our data suggests a role for astrocyte dysfunction in neurologic disease, and distinguish key regions of infection in the CNS for future investigations of CCHF.

Background: Human infection by orthopoxviruses is being reported with increasing frequency, attributed in part to the cessation of smallpox vaccination and concomitant waning of population-level immunity. In July 2015, a female resident of interior Alaska, presented to an urgent care clinic with a dermal lesion consistent with poxvirus infection. Laboratory testing of a virus isolated from the lesion confirmed infection by an Orthopoxvirus. Methods: The virus isolate was characterized by using electron microscopy and nucleic acid sequencing. An epidemiologic investigation that included patient interviews, contact tracing and serum testing, as well as environmental and small mammal sampling was conducted to identify the infection source and possible additional cases. Results: Neither signs of active infection nor evidence of recent prior infection were observed in any of the 4 patient contacts identified. The patient's infection source was not definitively identified. Potential routes of exposure included imported fomites from Azerbaijan by the patient's cohabiting partner, or from wild small mammals in or around the patient's residence. Phylogenetic analyses demonstrated that the virus represents a distinct and previously undescribed genetic lineage of Orthopoxvirus, which is most closely related to the Old World orthopoxviruses. Conclusions: Investigation findings point to infection of the patient following exposure in or near Fairbanks. This conclusion raises questions about the geographic origins (Old World versus North American) of the genus Orthopoxvirus. Clinicians should remain vigilant for signs of poxvirus infection and alert public health officials when cases are suspected.

In April 2014, a kidney transplant recipient in the United States experienced headache, diplopia, and confusion, followed by neurologic decline and death. An investigation to evaluate the possibility of donor-derived infection determined that 3 patients had received 4 organs (kidney, liver, heart/kidney) from the same donor. The liver recipient experienced tremor and gait instability; the heart/kidney and contralateral kidney recipients were hospitalized with encephalitis. None experienced gastrointestinal symptoms. Encephalitozoon cuniculi was detected by tissue PCR in the central nervous system of the deceased kidney recipient and in renal allograft tissue from both kidney recipients. Urine PCR was positive for E. cuniculi in the 2 surviving recipients. Donor serum was positive for E. cuniculi antibodies. E. cuniculi was transmitted to 3 recipients from 1 donor. This rare presentation of disseminated disease resulted in diagnostic delays. Clinicians should consider donor-derived microsporidial infection in organ recipients with unexplained encephalitis, even when gastrointestinal manifestations are absent.

Virus purification in a high-containment setting provides unique challenges due to barrier precautions and operational safety approaches that are not necessary in lower biosafety level (BSL) 2 environments. The need for high risk group pathogen diagnostic assay development, anti-viral research, pathogenesis and vaccine efficacy research necessitates work in BSL-3 and BSL-4 labs with infectious agents. When this work is performed in accordance with BSL-4 practices, modifications are often required in standard protocols. Classical virus purification techniques are difficult to execute in a BSL-3 or BSL-4 laboratory because of the work practices used in these environments. Orthopoxviruses are a family of viruses that, in some cases, requires work in a high-containment laboratory and due to size do not lend themselves to simpler purification methods. Current CDC purification techniques of orthopoxviruses uses 1,1,2-trichlorotrifluoroethane, commonly known as Genetron(R). Genetron(R) is a chlorofluorocarbon (CFC) that has been shown to be detrimental to the ozone and has been phased out and the limited amount of product makes it no longer a feasible option for poxvirus purification purposes. Here we demonstrate a new orthopoxvirus purification method that is suitable for high-containment laboratories and produces virus that is not only comparable to previous purification methods, but improves on purity and yield.

In 1973, investigators isolated a rickettsial organism, designated strain WB-8-2T, from an adult Amblyomma americanum tick collected at Land Between the Lakes National Recreation Area, TN, USA. This organism is now recognized as highly prevalent in A. americanum, as well as several other Amblyomma species found throughout the Western hemisphere. It has been suggested that cross-reactivity to WB-8-2T and similar strains contributes to the increasing number of spotted fever cases reported in the USA. In 1995, investigators provided preliminary evidence that this strain, as well as another strain from Missouri, represented a distinct taxonomic unit within the genus Rickettsia by evaluating sequences of the 16S rRNA and 17 kDa protein genes. However, the bacterium was never formally named, despite the use of the designation 'Rickettsia amblyommii' and later 'Candidatus Rickettsia amblyommii', for more than 20 years in the scientific literature. Herein, we provide additional molecular evidence to identify strain WB-8-2T as a representative strain of a unique rickettsial species and present a formal description for the species, with the proposed name modified to Rickettsia amblyommatis sp. nov. to conform to the International Code of Nomenclature of Prokaryotes. We also establish a pure culture of strain WB-8-2T and designate it as the type strain for the species. The type strain is WB-8-2T (=CRIRC RAM004T=CSURP2882T).

Vaccinia virus (VACV) has been implicated in infections of dairy cattle and humans, and outbreaks have substantially impacted local economies and public health in Brazil. During a 2005 outbreak, a VACV strain designated Serro 2 virus (S2V) was collected from a 30-year old male milker. Our aim was to phenotypically and genetically characterize this VACV Brazilian isolate. S2V produced small round plaques without associated comets when grown in BSC40 cells. Furthermore, S2V was less virulent than the prototype strain VACV-Western Reserve (WR) in a murine model of intradermal infection, producing a tiny lesion with virtually no surrounding inflammation. The genome of S2V was sequenced by primer walking. The coding region spans 184,572 bp and contains 211 predicted genes. Mutations in envelope genes specifically associated with small plaque phenotypes were not found in S2V; however, other alterations in amino acid sequences within these genes were identified. In addition, some immunomodulatory genes were truncated in S2V. Phylogenetic analysis using immune regulatory-related genes, besides the hemagglutinin gene, segregated the Brazilian viruses into two clusters, grouping the S2V into Brazilian VACV group 1. S2V is the first naturally-circulating human-associated VACV, with a low passage history, to be extensively genetically and phenotypically characterized.