High-throughput Sequencing to Identify Bacteremia in Children with Cancer.

Current methods to diagnose bacteremia are limited. In this pilot study of children with cancer presenting with fever, we determined the concordance between a novel high-throughput sequencing platform called BacCapSeq and blood culture. High-throughput sequencing had modest concordance with blood culture. Discordant organisms included those with both unlikely or potential clinical relevance.

Detection of antibodies to Anaplasma phagocytophilum and Babesia microti using linear peptides.

Anaplasma phagocytophilum and Babesia microti are emerging tick-borne pathogens in the United States. Although active infection is typically diagnosed by direct diagnostic tests, such as blood smear or polymerase chain reaction assay, serologic assays can be helpful to identify past infections, and the use of acute plus convalescent testing can potentially identify recent infections. We employed a peptide array to select sets of linear peptides for serologic diagnosis of infections with A. phagocytophilum and B. microti. Three optimal peptides were selected for each agent based on their performance with clinical specimens. All three A. phagocytophilum peptides were located within the conserved fragments of the MSP2 antigen. Two B. microti peptides were located in the N terminus of the SA-1 antigen; the third was in the BMN 1-17 antigen. We found that these peptides can be a useful tool for detection of antibody reactivity to both of these pathogens.

Human Polyomavirus 9-An Emerging Cutaneous and Pulmonary Pathogen in Solid Organ Transplant Recipients.

IMPORTANCE: We describe the first report to our knowledge of cutaneous and systemic pathogenicity of human polyomavirus 9 in solid organ transplant recipients. OBJECTIVE: Three solid organ transplant recipients developed a widespread, progressive, violaceous, and hyperkeratotic skin eruption. All died from pulmonary and multiorgan failure around 1 year from onset of the rash. Routine clinical diagnostic testing could not identify any causative agent; therefore, samples and autopsies were investigated for novel pathogens using high-throughput sequencing. DESIGN, SETTING, AND PARTICIPANTS: This case series, including 3 solid organ transplant recipients who developed characteristic pink, violaceous, or brown hyperkeratotic papules and plaques throughout the body, was conducted at the Columbia University Medical Center. Lesional skin biopsies were collected from all 3 patients and subjected to high-throughput illumina sequencing for identification of microbial pathogens. Human polyomavirus 9 was identified in lesional skin biopsies. We subsequently collected ocular swabs, oral swabs, urine samples, and blood samples from patients, and organ tissues at autopsy in 1 patient. We investigated these samples for the presence of human polyomavirus 9 using in situ hybridization and quantitative polymerase chain reaction (PCR) assays. MAIN OUTCOMES AND MEASURES: A description of the clinical and pathologic findings of 3 patients. RESULTS: This case series study found that human polyomavirus 9 was detected in the skin biopsies of all 3 patients by a capture-based high-throughput sequencing method platform (VirCapSeq-VERT). Human polyomavirus 9 was also detected in blood, oral, ocular swabs, and urine by real-time polymerase chain reaction (PCR) assay. In situ hybridization and quantitative PCR assays were performed on the skin biopsies from 3 patients and lung autopsy of 1 patient, which showed the presence of human polyomavirus 9 messenger RNA transcripts, indicating active viral replication and pathogenesis in the skin and lungs. CONCLUSIONS AND RELEVANCE: Human polyomavirus 9 was associated with the widespread cutaneous eruption. All 3 patients had progression of cutaneous disease, accompanied by clinical deterioration, pulmonary failure, and death. One patient underwent autopsy and human polyomavirus 9 was identified in the lungs and paratracheal soft tissue. These findings suggest that human polyomavirus 9 may be associated with cutaneous and possibly pulmonary infection and death in solid organ transplant recipients.

Vesicular Stomatitis Virus Chimeras Expressing the Oropouche Virus Glycoproteins Elicit Protective Immune Responses in Mice.

Oropouche virus (OROV) infection of humans is associated with a debilitating febrile illness that can progress to meningitis or encephalitis. First isolated from a forest worker in Trinidad and Tobago in 1955, the arbovirus OROV has since been detected throughout the Amazon basin with an estimated 500,000 human infections over 60 years. Like other members of the family Peribunyaviridae, the viral genome exists as 3 single-stranded negative-sense RNA segments. The medium-sized segment encodes a viral glycoprotein complex (GPC) that is proteolytically processed into two viral envelope proteins, Gn and Gc, responsible for attachment and membrane fusion. There are no therapeutics or vaccines to combat OROV infection, and we have little understanding of protective immunity to infection. Here, we generated a replication competent chimeric vesicular stomatitis virus (VSV), in which the endogenous glycoprotein was replaced by the GPC of OROV. Serum from mice immunized by intramuscular injection with VSV-OROV specifically neutralized wild-type OROV, and using peptide arrays we mapped multiple epitopes within an N-terminal variable region of Gc recognized by the immune sera. VSV-OROV lacking this variable region of Gc was also immunogenic in mice producing neutralizing sera that recognize additional regions of Gc. Challenge of both sets of immunized mice with wild-type OROV shows that the VSV-OROV chimeras reduce wild-type viral infection and suggest that antibodies that recognize the variable N terminus of Gc afford less protection than those that target more conserved regions of Gc. IMPORTANCE Oropouche virus (OROV), an orthobunyavirus found in Central and South America, is an emerging public health challenge that causes debilitating febrile illness. OROV is transmitted by arthropods, and increasing mobilization has the potential to significantly increase the spread of OROV globally. Despite this, no therapeutics or vaccines have been developed to combat infection. Using vesicular stomatitis (VSV) as a backbone, we developed a chimeric virus bearing the OROV glycoproteins (VSV-OROV) and tested its ability to elicit a neutralizing antibody response. Our results demonstrate that VSV-OROV produces a strong neutralizing antibody response that is at least partially targeted to the N-terminal region of Gc. Importantly, vaccination with VSV-OROV reduces viral loads in mice challenged with wild-type virus. These data provide novel evidence that targeting the OROV glycoproteins may be an effective vaccination strategy to combat OROV infection.

Immunoreactive peptide maps of SARS-CoV-2.

Serodiagnosis of SARS-CoV-2 infection is impeded by immunological cross-reactivity among the human coronaviruses (HCoVs): SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43, 229E, HKU1, and NL63. Here we report the identification of humoral immune responses to SARS-CoV-2 peptides that may enable discrimination between exposure to SARS-CoV-2 and other HCoVs. We used a high-density peptide microarray and plasma samples collected at two time points from 50 subjects with SARS-CoV-2 infection confirmed by qPCR, samples collected in 2004-2005 from 11 subjects with IgG antibodies to SARS-CoV-1, 11 subjects with IgG antibodies to other seasonal human coronaviruses (HCoV), and 10 healthy human subjects. Through statistical modeling with linear regression and multidimensional scaling we identified specific peptides that were reassembled to identify 29 linear SARS-CoV-2 epitopes that were immunoreactive with plasma from individuals who had asymptomatic, mild or severe SARS-CoV-2 infections. Larger studies will be required to determine whether these peptides may be useful in serodiagnostics.

Structural Basis and Evolution of Glycan Receptor Specificities within the Polyomavirus Family.

Asymptomatic infections with polyomaviruses in humans are common, but these small viruses can cause severe diseases in immunocompromised hosts. New Jersey polyomavirus (NJPyV) was identified via a muscle biopsy in an organ transplant recipient with systemic vasculitis, myositis, and retinal blindness, and human polyomavirus 12 (HPyV12) was detected in human liver tissue. The evolutionary origins and potential diseases are not well understood for either virus. In order to define their receptor engagement strategies, we first used nuclear magnetic resonance (NMR) spectroscopy to establish that the major capsid proteins (VP1) of both viruses bind to sialic acid in solution. We then solved crystal structures of NJPyV and HPyV12 VP1 alone and in complex with sialylated glycans. NJPyV employs a novel binding site for a α2,3-linked sialic acid, whereas HPyV12 engages terminal α2,3- or α2,6-linked sialic acids in an exposed site similar to that found in Trichodysplasia spinulosa-associated polyomavirus (TSPyV). Gangliosides or glycoproteins, featuring in mammals usually terminal sialic acids, are therefore receptor candidates for both viruses. Structural analyses show that the sialic acid-binding site of NJPyV is conserved in chimpanzee polyomavirus (ChPyV) and that the sialic acid-binding site of HPyV12 is widely used across the entire polyomavirus family, including mammalian and avian polyomaviruses. A comparison with other polyomavirus-receptor complex structures shows that their capsids have evolved to generate several physically distinct virus-specific receptor-binding sites that can all specifically engage sialylated glycans through a limited number of contacts. Small changes in each site may have enabled host-switching events during the evolution of polyomaviruses.IMPORTANCE Virus attachment to cell surface receptors is critical for productive infection. In this study, we have used a structure-based approach to investigate the cell surface recognition event for New Jersey polyomavirus (NJPyV) and human polyomavirus 12 (HPyV12). These viruses belong to the polyomavirus family, whose members target different tissues and hosts, including mammals, birds, fish, and invertebrates. Polyomaviruses are nonenveloped viruses, and the receptor-binding site is located in their capsid protein VP1. The NJPyV capsid features a novel sialic acid-binding site that is shifted in comparison to other structurally characterized polyomaviruses but shared with a closely related simian virus. In contrast, HPyV12 VP1 engages terminal sialic acids in a manner similar to the human Trichodysplasia spinulosa-associated polyomavirus. Our structure-based phylogenetic analysis highlights that even distantly related avian polyomaviruses possess the same exposed sialic acid-binding site. These findings complement phylogenetic models of host-virus codivergence and may also reflect past host-switching events.

A viral metagenomic survey identifies known and novel mammalian viruses in bats from Saudi Arabia.

Bats are implicated as natural reservoirs for a wide range of zoonotic viruses including SARS and MERS coronaviruses, Ebola, Marburg, Nipah, Hendra, Rabies and other lyssaviruses. Accordingly, many One Health surveillance and viral discovery programs have focused on bats. In this report we present viral metagenomic data from bats collected in the Kingdom of Saudi Arabia [KSA]. Unbiased high throughput sequencing of fecal samples from 72 bat individuals comprising four species; lesser mouse-tailed bat (Rhinopoma hardwickii), Egyptian tomb bat (Taphozous perforatus), straw-colored fruit bat (Eidolon helvum), and Egyptian fruit bat (Rousettus aegyptiacus) revealed molecular evidence of a diverse set of viral families: Picornaviridae (hepatovirus, teschovirus, parechovirus), Reoviridae (rotavirus), Polyomaviridae (polyomavirus), Papillomaviridae (papillomavirus), Astroviridae (astrovirus), Caliciviridae (sapovirus), Coronaviridae (coronavirus), Adenoviridae (adenovirus), Paramyxoviridae (paramyxovirus), and unassigned mononegavirales (chuvirus). Additionally, we discovered a bastro-like virus (Middle East Hepe-Astrovirus), with a genomic organization similar to Hepeviridae. However, since it shared homology with Hepeviridae and Astroviridae at ORF1 and in ORF2, respectively, the newly discovered Hepe-Astrovirus may represent a phylogenetic bridge between Hepeviridae and Astroviridae.

Fecal metagenomic profiles in subgroups of patients with myalgic encephalomyelitis/chronic fatigue syndrome.

BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by unexplained persistent fatigue, commonly accompanied by cognitive dysfunction, sleeping disturbances, orthostatic intolerance, fever, lymphadenopathy, and irritable bowel syndrome (IBS). The extent to which the gastrointestinal microbiome and peripheral inflammation are associated with ME/CFS remains unclear. We pursued rigorous clinical characterization, fecal bacterial metagenomics, and plasma immune molecule analyses in 50 ME/CFS patients and 50 healthy controls frequency-matched for age, sex, race/ethnicity, geographic site, and season of sampling. RESULTS: Topological analysis revealed associations between IBS co-morbidity, body mass index, fecal bacterial composition, and bacterial metabolic pathways but not plasma immune molecules. IBS co-morbidity was the strongest driving factor in the separation of topological networks based on bacterial profiles and metabolic pathways. Predictive selection models based on bacterial profiles supported findings from topological analyses indicating that ME/CFS subgroups, defined by IBS status, could be distinguished from control subjects with high predictive accuracy. Bacterial taxa predictive of ME/CFS patients with IBS were distinct from taxa associated with ME/CFS patients without IBS. Increased abundance of unclassified Alistipes and decreased Faecalibacterium emerged as the top biomarkers of ME/CFS with IBS; while increased unclassified Bacteroides abundance and decreased Bacteroides vulgatus were the top biomarkers of ME/CFS without IBS. Despite findings of differences in bacterial taxa and metabolic pathways defining ME/CFS subgroups, decreased metabolic pathways associated with unsaturated fatty acid biosynthesis and increased atrazine degradation pathways were independent of IBS co-morbidity. Increased vitamin B6 biosynthesis/salvage and pyrimidine ribonucleoside degradation were the top metabolic pathways in ME/CFS without IBS as well as in the total ME/CFS cohort. In ME/CFS subgroups, symptom severity measures including pain, fatigue, and reduced motivation were correlated with the abundance of distinct bacterial taxa and metabolic pathways. CONCLUSIONS: Independent of IBS, ME/CFS is associated with dysbiosis and distinct bacterial metabolic disturbances that may influence disease severity. However, our findings indicate that dysbiotic features that are uniquely ME/CFS-associated may be masked by disturbances arising from the high prevalence of IBS co-morbidity in ME/CFS. These insights may enable more accurate diagnosis and lead to insights that inform the development of specific therapeutic strategies in ME/CFS subgroups.

Identification of a novel polyomavirus in a pancreatic transplant recipient with retinal blindness and vasculitic myopathy.

BACKGROUND: A 33 year-old pancreatic transplant recipient developed weakness, retinal blindness, and necrotic plaques on her face, scalp, and hands. METHODS: A muscle biopsy was analyzed by light and electron microscopy and high-throughput nucleic acid sequencing. RESULTS: The biopsy revealed microthrombosis and viral particles in swollen endothelial cell nuclei. High-throughput sequencing of nucleic acid revealed a novel polyomavirus. In situ hybridization confirmed the presence of the polyomavirus in endothelial cells at sites of myositis and cutaneous necrosis. CONCLUSIONS: New Jersey polyomavirus (NJPyV-2013) is a novel polyomavirus that may have tropism for vascular endothelial cells.

Association of polymorphisms in the promoter regions of TNF-α (-308) with susceptibility to hepatitis E virus and TNF-α (-1031) and IFN-γ (+874) genes with clinical outcome of hepatitis E infection in India.

BACKGROUND & AIMS: Hepatitis E virus (HEV) is the predominant cause of acute viral hepatitis (AVH-E) and acute liver failure (ALF-E) among adults from developing countries. Pathogenesis of hepatitis E is poorly understood. Earlier, we showed association of elevated serum levels of TNF-α, IFN-γ, and IL-12 with ALF-E. The role of TNF-α and IFN-γ gene promoter polymorphisms with disease severity was investigated. METHODS: The study population included 374 anti-HEV negative apparently healthy controls, 136 subclinical hepatitis E, 353 AVH-E, and 25 ALF-E patients. Polymorphisms at promoter regions of TNF-α-308G/A, TNF-α-1031T/C, and IFN-γ+874T/A were investigated employing allelic discrimination/SNaPshot™ methods. RESULTS: ALF-E patients were younger with significantly higher ALT levels when compared to other categories. Genotype TNF-α-308AA frequency was significantly higher among subclinical and clinical hepatitis E than the controls (p=0.03, 0.0007). No significant difference was observed among AVH-E/ALF-E groups. The -308A allele was significantly higher in HEV-infected individuals; fatal ALF patients showed higher frequency than the recovered (p=0.024). TNF-α-1031CC, IFN-γ+874TT, and IFN-γ+874TA genotypes were significantly associated with clinical disease. With respect to the controls, genotype+874TA was more frequent in subclinical infection (p=0.005) while+874AA frequency was lower in the AVH-E category (p=0.003). CONCLUSIONS: The data reveal association of TNF-α-308AA genotype with susceptibility to HEV and that of TNF-α-1031CC and IFN-γ+874TT and TA with clinical disease, irrespective of the outcome. Higher -308A allele frequency was associated with susceptibility to HEV and the fatal outcome of ALF-E.