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Background Over 1 million Americans undergo joint replacement each year, and approximately 1 in 75 will incur a periprosthetic joint infection. Effective treatment necessitates pathogen identification, yet standard-of-care cultures fail to detect organisms in 10% to 20% of cases and require invasive sampling. We hypothesized that cell-free DNA (cfDNA) fragments from microorganisms in a periprosthetic joint infection can be found in the bloodstream and utilized to accurately identify pathogens via next-generation sequencing. Methods In this prospective observational study performed at a musculoskeletal specialty hospital in the U.S., we enrolled 53 adults with validated hip or knee periprosthetic joint infections. Participants had peripheral blood drawn immediately prior to surgical treatment. Microbial cfDNA from plasma was sequenced and aligned to a genome database with >1,000 microbial species. Intraoperative tissue and synovial fluid cultures were performed per the standard of care. The primary outcome was accuracy in organism identification with use of blood cfDNA sequencing, as measured by agreement with tissue-culture results. Results Intraoperative and preoperative joint cultures identified an organism in 46 (87%) of 53 patients. Microbial cfDNA sequencing identified the joint pathogen in 35 cases, including 4 of 7 culture-negative cases (57%). Thus, as an adjunct to cultures, cfDNA sequencing increased pathogen detection from 87% to 94%. The median time to species identification for cases with genus-only culture results was 3 days less than standard-of-care methods. Circulating cfDNA sequencing in 14 cases detected additional microorganisms not grown in cultures. At postoperative encounters, cfDNA sequencing demonstrated no detection or reduced levels of the infectious pathogen. Conclusions Microbial cfDNA from pathogens causing local periprosthetic joint infections can be detected in peripheral blood. These circulating biomarkers can be sequenced from noninvasive venipuncture, providing a novel source for joint pathogen identification. Further development as an adjunct to tissue cultures holds promise to increase the number of cases with accurate pathogen identification and improve time-to-speciation. This test may also offer a novel method to monitor infection clearance during the treatment period. Level of evidence Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Background Noninvasive diagnostic options are limited for invasive mold infections (IMIs). We evaluated the performance of a plasma microbial cell-free DNA sequencing (mcfDNA-Seq) test for diagnosing pulmonary IMI after hematopoietic cell transplant (HCT). Methods We retrospectively assessed the diagnostic performance of plasma mcfDNA-Seq next-generation sequencing in 114 HCT recipients with pneumonia after HCT who had stored plasma obtained within 14 days of diagnosis of proven/probable Aspergillus IMI (n = 51), proven/probable non-Aspergillus IMI (n = 24), possible IMI (n = 20), and non-IMI controls (n = 19). Sequences were aligned to a database including >400 fungi. Organisms above a fixed significance threshold were reported. Results Among 75 patients with proven/probable pulmonary IMI, mcfDNA-Seq detected ≥1 pathogenic mold in 38 patients (sensitivity, 51% [95% confidence interval {CI}, 39%–62%]). When restricted to samples obtained within 3 days of diagnosis, sensitivity increased to 61%. McfDNA-Seq had higher sensitivity for proven/probable non-Aspergillus IMI (sensitivity, 79% [95% CI, 56%–93%]) compared with Aspergillus IMI (sensitivity, 31% [95% CI, 19%–46%]). McfDNA-Seq also identified non-Aspergillus molds in an additional 7 patients in the Aspergillus subgroup and Aspergillus in 1 patient with possible IMI. Among 19 non-IMI pneumonia controls, mcfDNA-Seq was negative in all samples, suggesting a high specificity (95% CI, 82%–100%) and up to 100% positive predictive value (PPV) with estimated negative predictive values (NPVs) of 81%–99%. The mcfDNA-Seq assay was complementary to serum galactomannan index testing; in combination, they were positive in 84% of individuals with proven/probable pulmonary IMI. Conclusions Noninvasive mcfDNA-Seq had moderate sensitivity and high specificity, NPV, and PPV for pulmonary IMI after HCT, particularly for non-Aspergillus species.

This study sought to determine if atrial fibrillation (AF) ablation can be performed safely without bladder catheterization.Patients undergoing AF ablation often receive bladder catheters. Catheterization is associated with potential complications. The ABCD-AF (Avoiding Bladder Catheters During Atrial Fibrillation) ablation study evaluates the advantages of performing AF ablation without routine catheterization.In this single-center, prospective, randomized controlled trial, 80 patients received bladder catheterization (group A), and 80 patients received only as-needed catheterization (group B). The primary endpoint was a composite of cystitis, urethral injury, hematuria, dysuria, or urinary retention.The mean patient age was 63 ± 13 years, and 33% of patients were female. The primary outcome was reached in 45 patients in group A and 11 patients in group B (p 0.001). Urinary tract infection occurred in 7 patients in group A and 2 patients in group B (p = 0.17). Urinary retention occurred in 12 patients in group A and 5 patients in group B (p = 0.07). Randomization to catheterization carried an odds ratio of 8.1 (95% confidence interval [CI]: 3.7 to 17.5; p 0.001), and male sex carried an odds ratio of 3.8 (95% CI: 1.7 to 8.6; p = 0.001) for the primary endpoint. On subgroup analysis, randomization to undergo catheterization had no association with the primary outcome in female patients but had an odds ratio of 14.6 (95% CI: 5.6 to 38.1; p 0.001) in male patients. In multivariable analysis, sex and catheter status remained independently associated with the primary outcome.Bladder catheterization can be safely avoided in patients undergoing AF ablation and is associated with a significant reduction in adverse outcomes, especially in men.

Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use., This study describes a new method that improves the sensitivity of viral detection compared with next-generation sequencing and enables the detection of emerging flaviviruses not specifically targeted a priori. Metagenomic sequencing with spiked primer enrichment is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making it applicable for diagnostic laboratory and field use.

An 11-month-old male infant with ascending paralysis had an unremarkable initial cerebrospinal fluid (CSF) analysis and imaging. Progressive neurological symptoms resulted in repeated CSF sampling, microscopy, and plasma microbial cell-free DNA next-generation sequencing analysis, that in combination with epidemiology, confirmed the diagnosis.

Meningitis and encephalitis are leading causes of central nervous system (CNS) disease and often result in severe neurological compromise or death. Traditional diagnostic workflows largely rely on pathogen-specific tests, sometimes over days to weeks, whereas metagenomic next-generation sequencing (mNGS) profiles all nucleic acid in a sample. In this single-center, prospective study, 68 hospitalized patients with known (n = 44) or suspected (n = 24) CNS infections underwent mNGS from RNA and DNA to identify potential pathogens and also targeted sequencing of viruses using hybrid capture. Using a computational metagenomic classification pipeline based on KrakenUniq and BLAST, we detected pathogen nucleic acid in cerebrospinal fluid (CSF) from 22 subjects, 3 of whom had no clinical diagnosis by routine workup. Among subjects diagnosed with infection by serology and/or peripheral samples, we demonstrated the utility of mNGS to detect pathogen nucleic acid in CSF, importantly for the Ixodes scapularis tick-borne pathogens Powassan virus, Borrelia burgdorferi, and Anaplasma phagocytophilum. We also evaluated two methods to enhance the detection of viral nucleic acid, hybrid capture and methylated DNA depletion. Hybrid capture nearly universally increased viral read recovery. Although results for methylated DNA depletion were mixed, it allowed the detection of varicella-zoster virus DNA in two samples that were negative by standard mNGS. Overall, mNGS is a promising approach that can test for multiple pathogens simultaneously, with efficacy similar to that of pathogen-specific tests, and can uncover geographically relevant infectious CNS disease, such as tick-borne infections in New England. With further laboratory and computational enhancements, mNGS may become a mainstay of workup for encephalitis and meningitis. IMPORTANCE Meningitis and encephalitis are leading global causes of central nervous system (CNS) disability and mortality. Current diagnostic workflows remain inefficient, requiring costly pathogen-specific assays and sometimes invasive surgical procedures. Despite intensive diagnostic efforts, 40 to 60% of people with meningitis or encephalitis have no clear cause of CNS disease identified. As diagnostic uncertainty often leads to costly inappropriate therapies, the need for novel pathogen detection methods is paramount. Metagenomic next-generation sequencing (mNGS) offers the unique opportunity to circumvent these challenges using unbiased laboratory and computational methods. Here, we performed comprehensive mNGS from 68 prospectively enrolled patients with known (n = 44) or suspected (n = 24) CNS viral infection from a single center in New England and evaluated enhanced methods to improve the detection of CNS pathogens, including those not traditionally identified in the CNS by nucleic acid detection. Overall, our work helps elucidate how mNGS can become integrated into the diagnostic toolkit for CNS infections.

Host inflammatory responses predict worse outcome in severe pneumonia, yet little is known about what drives dysregulated inflammation. We performed metagenomic sequencing of microbial cell-free DNA (mcfDNA) in 83 mechanically ventilated patients (26 culture-positive, 41 culture-negative pneumonia, 16 uninfected controls). Culture-positive patients had higher levels of mcfDNA than those with culture-negative pneumonia and uninfected controls (p

Background Microbial cell-free DNA (mcfDNA) sequencing of plasma can identify the presence of a pathogen in a host. In this study, we evaluated the duration of pathogen detection by mcfDNA sequencing vs conventional blood culture in patients with bacteremia. Methods Blood samples from patients with culture-confirmed bloodstream infection were collected within 24 hours of the index positive blood culture and 48 to 72 hours thereafter. mcfDNA was extracted from plasma, and next-generation sequencing was applied. Reads were aligned against a curated pathogen database. Statistical significance was defined with Bonferroni adjustment for multiple comparisons (P Results A total of 175 patients with Staphylococcus aureus bacteremia (n = 66), gram-negative bacteremia (n = 74), or noninfected controls (n = 35) were enrolled. The overall sensitivity of mcfDNA sequencing compared with index blood culture was 89.3% (125 of 140), and the specificity was 74.3%. Among patients with bacteremia, pathogen-specific mcfDNA remained detectable for significantly longer than conventional blood cultures (median 15 days vs 2 days; P Conclusions Pathogen mcfDNA identified the bacterial etiology of bloodstream infection for a significantly longer interval than conventional cultures, and its duration of detection was associated with increased risk for metastatic infection. mcfDNA could play a role in the diagnosis of partially treated endovascular infections.

Background Laboratory confirmation of early Lyme borreliosis (LB) is challenging. Serology is insensitive during the first days to weeks of infection, and blood polymerase chain reaction (PCR) offers similarly poor performance. Here, we demonstrate that detection of Borrelia burgdorferi (B.b.) cell-free DNA (cfDNA) in plasma can improve diagnosis of early LB. Methods B.b. detection in plasma samples using unbiased metagenomic cfDNA sequencing performed by a commercial laboratory (Karius Inc) was compared with serology and blood PCR in 40 patients with physician-diagnosed erythema migrans (EM), 28 of whom were confirmed to have LB by skin biopsy culture (n = 18), seroconversion (n = 2), or both (n = 8). B.b. sequence analysis was performed using investigational detection thresholds, different from Karius’ clinical test. Results B.b. cfDNA was detected in 18 of 28 patients (64%) with laboratory-confirmed EM. In comparison, sensitivity of acute-phase serology using modified 2-tiered testing (MTTT) was 50% (P = .45); sensitivity of blood PCR was 7% (P = .0002). Combining B.b. cfDNA detection and MTTT increased diagnostic sensitivity to 86%, significantly higher than either approach alone (P ≤ .04). B.b. cfDNA sequences matched precisely with strain-specific sequence generated from the same individual’s cultured B.b. isolate. B.b. cfDNA was not observed at any level in plasma from 684 asymptomatic ambulatory individuals. Among 3000 hospitalized patients tested as part of clinical care, B.b. cfDNA was detected in only 2 individuals, both of whom had clinical presentations consistent with LB. Conclusions This is the first report of B.b. cfDNA detection in early LB and a demonstration of potential diagnostic utility. The combination of B.b. cfDNA detection and acute-phase MTTT improves clinical sensitivity for diagnosis of early LB.

Progressive multifocal leukoencephalopathy is a rare, demyelinating disease of the central nervous system caused by JC virus. Fewer than 30 cases have been reported in HIV- and non-infected children. We report the case of a 15-year-old girl with progressive multifocal leukoencephalopathy and AIDS who presented with nystagmus, dysarthria and ataxia. Following combined antiretroviral therapy, she developed immune reconstitution inflammatory syndrome, which proved fatal.

Background: Cell-free DNA (cfDNA) sequencing has emerged as an effective laboratory method for rapid and noninvasive diagnosis in prenatal screening testing, organ transplant rejection screening, and oncology liquid biopsies but clinical experience for use of this technology in diagnostic evaluation of infections in immunocompromised hosts is limited. Methods: We conducted an exploratory study using next-generation sequencing (NGS) for detection of microbial cfDNA in a cohort of ten immunocompromised patients with febrile neutropenia, pneumonia or intra-abdominal infection. Results: Pathogen identification by cfDNA NGS demonstrated positive agreement with conventional diagnostic laboratory methods in 7 (70%) cases, including patients with proven/probable invasive aspergillosis, Pneumocystis jirovecii pneumonia, Stenotrophomonas maltophilia bacteremia, Cytomegalovirus and Adenovirus viremia. NGS results were discordant in 3 (30%) cases including two patients with culture negative sepsis who had undergone hematopoietic stem cell transplant in whom cfDNA testing identified the potential etiological agent of sepsis; and one kidney transplant recipient with invasive aspergillosis who had received >6 months of antifungal therapy prior to NGS testing. Conclusion: These observations support the clinical utility of measurement of microbial cfDNA sequencing from peripheral blood for rapid noninvasive diagnosis of infections in immunocompromised hosts. Larger studies are needed.

We analyzed characteristics of 86 Group A streptococcal bacteremia cases at Boston Children's Hospital from 1992 to 2012. Twenty-three percent of children had severe disease, using intensive care unit admission (18), disability (7) or death (2) as indicators. Children with bacteremia without a source (30% of cases) were less likely to have severe disease than children with focal infections in adjusted models.

A short prodrome in children is associated with more severe disease and increased risk for death., We describe the clinical, laboratory, and radiographic characteristics of 15 cases of eastern equine encephalitis in children during 1970–2010. The most common clinical and laboratory features were fever, headache, seizures, peripheral leukocytosis, and cerebrospinal fluid neutrophilic pleocytosis. Radiographic lesions were found in the basal ganglia, thalami, and cerebral cortex. Clinical outcomes included severe neurologic deficits in 5 (33%) patients, death of 4 (27%), full recovery of 4 (27%), and mild neurologic deficits in 2 (13%). We identify an association between a short prodrome and an increased risk for death or for severe disease.

The discovery of African henipaviruses (HNVs) related to pathogenic Hendra virus (HeV) and Nipah virus (NiV) from Southeast Asia and Australia presents an open-ended health risk. Cell receptor use by emerging African HNVs at the stage of host-cell entry is a key parameter when considering the potential for spillover and infection of human populations. The attachment glycoprotein from a Ghanaian bat isolate (GhV-G) exhibits30% sequence identity with Asiatic NiV-G/HeV-G. Here, through functional and structural analysis of GhV-G, we show how this African HNV targets the same human cell-surface receptor (ephrinB2) as the Asiatic HNVs. We first characterized this virus-receptor interaction crystallographically. Compared with extant HNV-G-ephrinB2 structures, there was significant structural variation in the six-bladed β-propeller scaffold of the GhV-G receptor-binding domain, but not the Greek key fold of the bound ephrinB2. Analysis revealed a surprisingly conserved mode of ephrinB2 interaction that reflects an ongoing evolutionary constraint among geographically distal and phylogenetically divergent HNVs to maintain the functionality of ephrinB2 recognition during virus-host entry. Interestingly, unlike NiV-G/HeV-G, we could not detect binding of GhV-G to ephrinB3. Comparative structure-function analysis further revealed several distinguishing features of HNV-G function: a secondary ephrinB2 interaction site that contributes to more efficient ephrinB2-mediated entry in NiV-G relative to GhV-G and cognate residues at the very C terminus of GhV-G (absent in Asiatic HNV-Gs) that are vital for efficient receptor-induced fusion, but not receptor binding per se. These data provide molecular-level details for evaluating the likelihood of African HNVs to spill over into human populations.

West Nile virus is a mosquito-borne, neurotropic flavivirus that causes encephalitis in humans and animals. Since being introduced into the Western hemisphere in 1999, WNV has spread rapidly across North America, identifying this virus as an important emerging pathogen. In this study, we developed a DNA-launched infectious molecular clone of WNV that encodes a GFP reporter gene. Transfection of cells with the plasmid encoding this recombinant virus (pWNII-GFP) resulted in the production of infectious WNV capable of expressing GFP at high levels shortly after infection of a variety of cell types, including primary neurons and dendritic cells. Infection of cells with WNII-GFP virus was productive, and could be inhibited with both monoclonal antibodies and interferon-β, highlighting the potential of this system in the development and characterization of novel inhibitors and therapeutics for WNV infection. As expected, insertion of the reporter gene into the viral genome was associated with a reduced rate of viral replication, providing the selective pressure for the development of variants that no longer encoded the full-length reporter gene cassette. We anticipate this DNA-based, infectious WNV reporter virus will allow novel approaches for the study of WNV infection and its inhibition both in vitro and in vivo.

To the Editor: Coxsackievirus A6 (CVA6) is a human enterovirus associated with herpangina in infants. In the winter of 2012, we evaluated a cluster of 8 patients, 4 months–3 years of age, who were brought for treatment at Boston Children’s Hospital (Boston, MA, USA) with a variant of hand, foot, and mouth disease (HFMD) that has now been linked to CVA6 (Table). During this same period, the Boston Public Health Commission’s syndromic surveillance system detected a 3.3-fold increase in emergency department discharge diagnoses of HFMD. In the United States, HFMD typically occurs in the summer and early autumn and is characterized by a febrile enanthem of oral ulcers and macular or vesicular lesions on the palms and soles; the etiologic agents are most often CVA16 and enterovirus 71. Table Demographic and clinical characteristics of patients with CVA6-associated HFMD, Boston, Massachusetts, USA, 2012* In contrast to the typical manifestation, the patients in the Boston cluster exhibited symptoms in late winter (Table) and had perioral (Figure, panel A) and perirectal (Figure, panel B) papules and vesicles on the dorsal aspects of the hands and feet (Figure, panel C). Patients experienced a prodrome lasting 1–3 days, consisting of fever (8 patients), upper respiratory tract symptoms (4 patients), and irritability (7 patients). This prodrome was followed by the development of a perioral papular rash (8 patients), which was often impetiginized with secondary crusting; a prominent papulovesicular rash on the dorsum of the hands and feet (6 patients); and a perirectal eruption (7 patients). Half of the patients had intraoral lesions. Fever abated in most of the patients within a day after onset of the exanthem. The rash resolved over 7–14 days with no residual scarring. Samples from the oropharynx, rectum, and vesicles from these patients were sent to the Centers for Disease Control and Prevention (Atlanta, GA, USA) for analysis. Reverse transcription PCR and sequencing by using primers specific for a portion of the viral protein 1 coding region identified CVA6 (1) (Table). Figure Manifestations of hand, foot, and mouth disease in patients, Boston, Massachusetts, USA, 2012. Discrete superficial crusted erosions and vesicles symmetrically distributed in the perioral region (A), in the perianal region (B), and on the dorsum of the ... Outbreaks of HFMD caused by CVA6 have been described in Singapore, Finland, Taiwan, and most recently in Japan; most cases have occurred in the warmer months (2–6). Cases in the cluster described here are likely related to an emerging outbreak of CVA6-associated HFMD in the United States (7). The atypical seasonality of the outbreak, during the winter in Boston, could be related to the unusually mild temperatures in the winter of 2012. Recent CVA6 outbreaks have been characterized by a febrile illness associated with an oral enanthem and lesions on the palms, soles, and buttocks. CVA6 infections in Taiwan during 2004–2009 were associated with HFMD in 13% of cases, with disease defined as oral ulcers on the tongue or buccal mucosa and vesicular rashes on the palms, soles, knees, or buttocks (2). In Singapore, where CVA6 accounted for 24% of HFMD cases, patients had oral lesions and

The HIV-1 envelope glycoprotein is a trimeric complex of heterodimers composed of a surface glycoprotein, gp120, and a transmembrane component, gp41. The association of this complex with CD4 stabilizes the coreceptor-binding site of gp120 and promotes the exposure of the gp41 helical region 1 (HR1). Here, we show that a 15-amino-acid peptide mimetic of the HIV-1 coreceptor CCR5 fused to a dimeric antibody Fc domain (CCR5mim-Ig) bound two gp120 molecules per envelope glycoprotein complex and by itself promoted HR1 exposure. CCR5mim-Ig also stabilized the association of a CD4-mimetic peptide with the envelope glycoprotein. A fusion of the CD4- and CCR5-mimetic peptides, DM1, bound gp120 and neutralized R5, R5X4, and X4 HIV-1 isolates comparably to CD4, and they did so markedly more efficiently than either peptide alone. Our data indicate that the potency of DM1-Ig derives from its avidity for the HIV-1 envelope glycoprotein trimer and from the bidirectional induction of its receptor-mimetic components. DM1 has significant advantages over other inhibitors that target both coreceptor and CD4-binding sites, and it may serve as a lead for a new class of HIV-1 inhibitor peptides.

Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3) are recently identified viral restriction factors that inhibit infection mediated by the influenza A virus (IAV) hemagglutinin (HA) protein. Here we show that IFITM proteins restricted infection mediated by the entry glycoproteins (GP1,2) of Marburg and Ebola filoviruses (MARV, EBOV). Consistent with these observations, interferon-β specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV. We observed distinct patterns of IFITM-mediated restriction: compared with IAV, the entry processes of MARV and EBOV were less restricted by IFITM3, but more restricted by IFITM1. Moreover, murine Ifitm5 and 6 did not restrict IAV, but efficiently inhibited filovirus entry. We further demonstrate that replication of infectious SARS coronavirus (SARS-CoV) and entry mediated by the SARS-CoV spike (S) protein are restricted by IFITM proteins. The profile of IFITM-mediated restriction of SARS-CoV was more similar to that of filoviruses than to IAV. Trypsin treatment of receptor-associated SARS-CoV pseudovirions, which bypasses their dependence on lysosomal cathepsin L, also bypassed IFITM-mediated restriction. However, IFITM proteins did not reduce cellular cathepsin activity or limit access of virions to acidic intracellular compartments. Our data indicate that IFITM-mediated restriction is localized to a late stage in the endocytic pathway. They further show that IFITM proteins differentially restrict the entry of a broad range of enveloped viruses, and modulate cellular tropism independently of viral receptor expression., Author Summary Cells express restriction factors, proteins whose primary activity is to inhibit viral replication. We have recently described a family of restriction factors, interferon-inducible transmembrane (IFITM) proteins, that interfere with replication of influenza A virus. The IFITM proteins uniquely inhibit replication early in the viral life-cycle, before the virus can successfully enter the cell cytoplasm. Here we show that the entry processes of several highly pathogenic viruses – Marburg virus, Ebola virus, and SARS coronavirus – are similarly disrupted by IFITM proteins. We compared IFITM-mediated restriction of these viruses with influenza A virus, and discovered that individual IFITM proteins are specialized for restriction. For example, we describe two mouse IFITM proteins that efficiently restrict entry of Marburg and Ebola viruses, but which do not inhibit influenza A virus. We further show that we can circumvent IFITM-mediated restriction by inducing a virus to enter a cell at or near the plasma membrane. This observation indicates that restriction is not a global property of the cell, but rather is localized to late endosomal and lysosomal compartments, the usual entry sites of IFITM-restricted viruses. This study therefore enhances our understanding of how the innate immune system controls influenza A virus and other pathogenic viruses.

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease in humans with high rates of mortality. The virus has a tripartite genome composed of a small (S), a medium (M) and a large (L) RNA segment; the M segment encodes the two viral glycoproteins, GN and GC. Whilst relatively few full-length M segment sequences are available, it is apparent that both GN and GC may exhibit significant sequence diversity. It is unknown whether considerable antigenic differences exist between divergent CCHFV strains, or whether there are conserved neutralizing epitopes. The M segments derived from viral isolates of a human case of CCHF in South Africa (SPU 41/84), an infected tick (Hyalomma marginatum) in South Africa (SPU 128/81), a human case in Congo (UG 3010), an infected individual in Uzbekistan (U2-2-002) and an infected tick (Hyalomma asiaticum) in China (Hy13) were sequenced fully, and the glycoproteins were expressed. These novel sequences showed high variability in the N-terminal region of GN and more modest differences in the remainder of GN and in GC. Phylogenetic analyses placed these newly identified strains in three of the four previously described M segment groups. Studies with a panel of mAbs specific to GN and GC indicated that there were significant antigenic differences between the M segment groups, although several neutralizing epitopes in both GN and GC were conserved among all strains examined. Thus, the genetic diversity exhibited by CCHFV strains results in significant antigenic differences that will need to be taken into consideration for vaccine development.

West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation on flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. Therefore, we examined the impact of prM and E glycosylation on WNV assembly and infectivity. Similar to other flaviviruses, expression of WNV prM and E resulted in the release of subviral particles (SVPs). Removing the prM glycosylation site in a lineage I or II strain decreased SVP release, as did removal of the glycosylation site in a lineage I E protein. Addition of the E protein glycosylation site in a lineage II strain that lacked this site increased SVP production. Similar results were obtained in the context of either reporter virus particles (RVPs) or infectious lineage II WNV. RVPs or virions bearing combinations of glycosylated and nonglycosylated forms of prM and E could infect mammalian, avian, and mosquito cells (BHK-21, QT6, and C6/36, respectively). Those particles lacking glycosylation on the E protein were modestly more infectious per genome copy on BHK-21 and QT6 cells, while this absence greatly enhanced the infection of C6/36 cells. Thus, glycosylation of WNV prM and E proteins can affect the efficiency of virus release and infection in a manner that is cell type and perhaps species dependent. This suggests a multifaceted role for envelope N-linked glycosylation in WNV biology and tropism.

This is a report of an 11-year-old, prepubertal boy with acute-onset urinary urgency and frequency, obsessions and compulsions related to urination, severe mood lability, inattention, impulsivity, hyperactivity, and intermittent periods of immobilization. Fever, cough, otitis, and sinusitis preceded neuropsychiatric symptoms. Antistreptolysin O and DNAse B antibody titers were elevated, and magnetic resonance imaging revealed bilateral diffuse caudate nuclei swelling. Plasmapheresis resulted in significant and rapid clinical improvement of obsessive-compulsive disorder symptoms and a simultaneous decrease in basal ganglia swelling, consistent with an immune-mediated pathophysiological process involving group A beta-hemolytic streptococci. Hyperactivity, impulsivity, and inattention improved with lorazepam, suggesting that the attention-deficit/hyperactivity disorder symptoms could be manifestations of catatonia.

West Nile virus (WNV) is a neurotropic flavivirus within the Japanese encephalitis antigenic complex that is responsible for causing West Nile encephalitis in humans. The surface of WNV virions is covered by a highly ordered icosahedral array of envelope proteins that is responsible for mediating attachment and fusion with target cells. These envelope proteins are also primary targets for the generation of neutralizing antibodies in vivo. In this study, we describe a novel approach for measuring antibody-mediated neutralization of WNV infection using virus-like particles that measure infection as a function of reporter gene expression. These reporter virus particles (RVPs) are produced by complementation of a sub-genomic replicon with WNV structural proteins provided in trans using conventional DNA expression vectors. The precision and accuracy of this approach stem from an ability to measure the outcome of the interaction between antibody and viral antigens under conditions that satisfy the assumptions of the law of mass action as applied to virus neutralization. In addition to its quantitative strengths, this approach allows the production of WNV RVPs bearing the prM-E proteins of different WNV strains and mutants, offering considerable flexibility for the study of the humoral immune response to WNV in vitro. WNV RVPs are capable of only a single round of infection, can be used under BSL-2 conditions, and offer a rapid and quantitative approach for detecting virus entry and its inhibition by neutralizing antibody.

Human T-cell Immunoglobulin and Mucin-domain containing proteins (TIM1, 3, and 4) specifically bind phosphatidylserine (PS). TIM1 has been proposed to serve as a cellular receptor for hepatitis A virus and Ebola virus and as an entry factor for dengue virus. Here we show that TIM1 promotes infection of retroviruses and virus-like particles (VLPs) pseudotyped with a range of viral entry proteins, in particular those from the filovirus, flavivirus, New World arenavirus and alphavirus families. TIM1 also robustly enhanced the infection of replication-competent viruses from the same families, including dengue, Tacaribe, Sindbis and Ross River viruses. All interactions between TIM1 and pseudoviruses or VLPs were PS-mediated, as demonstrated with liposome blocking and TIM1 mutagenesis experiments. In addition, other PS-binding proteins, such as Axl and TIM4, promoted infection similarly to TIM1. Finally, the blocking of PS receptors on macrophages inhibited the entry of Ebola VLPs, suggesting that PS receptors can contribute to infection in physiologically relevant cells. Notably, infection mediated by the entry proteins of Lassa fever virus, influenza A virus and SARS coronavirus was largely unaffected by TIM1 expression. Taken together our data show that TIM1 and related PS-binding proteins promote infection of diverse families of enveloped viruses, and may therefore be useful targets for broad-spectrum antiviral therapies., Author Summary To infect cells, enveloped viruses typically utilize cellular receptors, which mediate specific, high-affinity interactions with the viral entry protein and prime the entry protein for subsequent steps in the viral entry process. Viral entry is also enhanced by attachment factors. Although less specific than receptors, attachment factors can alter the course of infection and thus severity of viral disease by increasing the infection efficiency of specific target cells. Here we observed that TIM proteins, a group of proteins that promote phagocytosis of apoptotic cells, can dramatically enhance the entry of a number of viruses, including Ebola, West Nile and dengue viruses, whereas they have little effect on the entry of other viruses. The inability of a virus to use TIM proteins may be due to the presence of an abundant, high-affinity receptor (Lassa fever virus), or because the TIM proteins direct virions to a non-productive internalization pathway (SARS coronavirus, influenza A virus). Mechanistically, TIM proteins appear to interact with enveloped viruses and apoptotic cells similarly by binding phosphatidylserine residues exposed on the viral and cellular membranes. Collectively our studies show that TIM proteins are attachment factors that can substantially improve the infection efficiency of a number of pathogenic viruses.