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Cytomegalovirus (CMV) is a beta-herpes virus causing common infections, often asymptomatic in healthy individuals. However, it poses increased risks to immunocompromised individuals and can cause congenital infections, leading to severe disabilities. CMV infection can cause significant hematological abnormality. A four-month-old female infant was admitted for decreased feeding for two days. She was severely pale, without hepatosplenomegaly. In initial laboratory investigations hemoglobin, platelet count, and white blood cells were decreased. The patient was transfused with whole blood and referred to a tertiary care center. Further workup, including bone marrow biopsy, showed hypocellular marrow. The Urine CMV Polymerase Chain Reaction (PCR) test returned highly positive with a viral load of 1,700,000 copies/mL. This patient was diagnosed with CMV-associated bone marrow suppression, and she was prescribed valganciclovir at a dosage of 16 mg/kg/dose every 12 h for 6 months. She had shown significant hematologic parameter improvement during subsequent follow-up. Pancytopenia in infancy should include a differential diagnosis for CMV infection. The early recognition and correct infection management, including antiviral therapy and symptomatic treatment, yield a better prognosis., Competing Interests: Declarations Ethics approval and consent to participate Our institution does not require ethical approval for case studies. The patient’s parent’s consent was obtained. Consent for publication Written Informed consent for publication of clinical details and clinical images was obtained from the patient's parents. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

There is increasing evidence that thirdhand exposure to e-cigarette vapor (e-vapor) can have detrimental effects on the lungs. However, whether maternal exposure during pregnancy results in harmful changes to the offspring is unknown. Using two different e-cigarette settings (low vs. high power), BALB/c mice were subjected to thirdhand e-vapor (e-vapor deposited onto towels, towels changed daily) in the absence or presence of nicotine, before, during, and after pregnancy. Male adult offspring were then infected with mouse-adapted influenza A virus (A/PR/8/34 H1N1; Flu) and lung and bone marrow immune cell responses were assessed 7 days postinfection. Maternal thirdhand exposure to low-power ( M LP) or high-power ( M HP) e-vapor with nicotine ( M LP + NIC and M HP + NIC, respectively) increased the percentage of lung immune cells and neutrophils in the bone marrow. Interestingly, Flu-infected offspring from M LP + NIC and M HP + NIC groups had lower percentages of lung alveolar macrophages and more pronounced increases in neutrophils in the bone marrow, when compared with offspring from M Sham Flu controls. Flu infection also decreased the percentage of lung CD4+ T cells and increased the percentage of lung CD8+ T cells, irrespective of maternal exposure ( M LP -/+ NIC and M HP -/+ NIC). Significantly, both M LP + NIC and M HP + NIC resulted in blunted activation of lung CD4+ T cells, but only M LP + NIC caused blunted activation of lung CD8+ T cells. Together, we show for the first time that maternal thirdhand exposure to e-vapor results in significant, long-lived effects on lung and bone marrow immune cell responses in offspring at baseline and response to Flu infection. NEW & NOTEWORTHY Maternal exposure to environmental residues of e-cigarette use has significant effects on immune cell responses in the lungs and bone marrow of offspring at both baseline and in response to influenza A virus (Flu) infection.

Feline leukemia virus (FeLV) is a highly debilitating cat pathogen due to its ability to cause many pathological changes. Therefore, identifying the virus directly in bone marrow can be a highly relevant diagnostic tool even in the absence of viraemia. The aim of this study was to compare the diagnostic efficiency of immunocytochemistry (ICC) of bone marrow aspirates with enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Blood samples were collected from 188 cats and separated into aliquots of whole blood for nested PCR using the U3 LTR region and the gag gene of FeLV-A as reference and serum for detection of the p27 antigen by ELISA. Bone marrow samples from these cats were placed on silanized slides for anti-FeLV ICC using gp70 as primary antibody. A total of 28.2% of the cats tested for FeLV were positive in at least one of the tests, with 26.6% positive by PCR, 18.1% by ICC and 11.2% by ELISA. Cohen's kappa agreement test revealed moderate agreement between ELISA and PCR results and substantial agreement between ICC and ELISA and between ICC and PCR. The results indicated that ICC of bone marrow is an efficient novel diagnostic test for FeLV infection., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In vitro studies have shown that deletion of nef and deleterious mutation in the Nef dimerization interface attenuates HIV replication and associated pathogenesis. Humanized rodents with human immune cells and lymphoid tissues are robust in vivo models for investigating the interactions between HIV and the human immune system. Here, we demonstrate that nef deletion impairs HIV replication and HIV-induced immune dysregulation in the blood and human secondary lymphoid tissue (human spleen) in bone marrow-liver-thymus-spleen (BLTS) humanized mice. Furthermore, we also show that nef defects (via deleterious mutations in the dimerization interface) impair HIV replication and HIV-induced immune dysregulation in the blood and human spleen in BLTS-humanized mice. We demonstrate that the reduced replication of nef-deleted and nef-defective HIV is associated with robust antiviral innate immune response, and T helper 1 response. Our results support the proposition that Nef may be a therapeutic target for adjuvants in HIV cure strategies., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hematopoiesis is a tightly regulated process that gets skewed toward myelopoiesis. This restrains lymphopoiesis, but the role of lymphocytes in this process is not well defined. To unravel the intricacies of neutrophil responses in COVID-19, we performed bulk RNAseq on neutrophils from healthy controls and COVID-19 patients. Principal component analysis revealed distinguishing neutrophil gene expression alterations in COVID-19 patients. ICU and ward patients displayed substantial transcriptional changes, with ICU patients exhibiting a more pronounced response. Intriguingly, neutrophils from COVID-19 patients, notably ICU patients, exhibited an enrichment of immunoglobulin (Ig) and B cell lineage-associated genes, suggesting potential lineage plasticity. We validated our RNAseq findings in a larger cohort. Moreover, by reanalyzing single-cell RNA sequencing (scRNAseq) data on human bone marrow (BM) granulocytes, we identified the cluster of granulocyte-monocyte progenitors (GMP) enriched with Ig and B cell lineage-associated genes. These cells with lineage plasticity may serve as a resource depending on the host's needs during severe systemic infection. This distinct B cell subset may play a pivotal role in promoting myelopoiesis in response to infection. The scRNAseq analysis of BM neutrophils in infected mice further supported our observations in humans. Finally, our studies using an animal model of acute infection implicate IL-7/GM-CSF in influencing neutrophil and B cell dynamics. Elevated GM-CSF and reduced IL-7 receptor expression in COVID-19 patients imply altered hematopoiesis favoring myeloid cells over B cells. Our findings provide novel insights into the relationship between the B-neutrophil lineages during severe infection, hinting at potential implications for disease pathogenesis., Importance: This study investigates the dynamics of hematopoiesis in COVID-19, focusing on neutrophil responses. Through RNA sequencing of neutrophils from healthy controls and COVID-19 patients, distinct gene expression alterations are identified, particularly in ICU patients. Notably, neutrophils from COVID-19 patients, especially in the ICU, exhibit enrichment of immunoglobulin and B cell lineage-associated genes, suggesting potential lineage plasticity. Validation in a larger patient cohort and single-cell analysis of bone marrow granulocytes support the presence of granulocyte-monocyte progenitors with B cell lineage-associated genes. The findings propose a link between B-neutrophil lineages during severe infection, implicating a potential role for these cells in altered hematopoiesis favoring myeloid cells over B cells. Elevated GM-CSF and reduced IL-7 receptor expression in stress hematopoiesis suggest cytokine involvement in these dynamics, providing novel insights into disease pathogenesis., Competing Interests: The authors declare no conflict of interest.

Generation of a stable long-lived plasma cell (LLPC) population is the sine qua non of durable antibody responses after vaccination or infection. We studied 20 individuals with a prior coronavirus disease 2019 infection and characterized the antibody response using bone marrow aspiration and plasma samples. We noted deficient generation of spike-specific LLPCs in the bone marrow after severe acute respiratory syndrome coronavirus 2 infection. Furthermore, while the regression model explained 98% of the observed variance in anti-tetanus immunoglobulin G levels based on LLPC enzyme-linked immunospot assay, we were unable to fit the same model with anti-spike antibodies, again pointing to the lack of LLPC contribution to circulating anti-spike antibodies., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

HIV is difficult to eradicate due to the persistence of a long-lived reservoir of latently infected cells. Previous studies have shown that natural killer cells are important to inhibiting HIV infection, but it is unclear whether the administration of natural killer cells can reduce rebound viremia when anti-retroviral therapy is discontinued. Here we show the administration of allogeneic human peripheral blood natural killer cells delays viral rebound following interruption of anti-retroviral therapy in humanized mice infected with HIV-1. Utilizing genetically barcoded virus technology, we show these natural killer cells efficiently reduced viral clones rebounding from latency. Moreover, a kick and kill strategy comprised of the protein kinase C modulator and latency reversing agent SUW133 and allogeneic human peripheral blood natural killer cells during anti-retroviral therapy eliminated the viral reservoir in a subset of mice. Therefore, combinations utilizing latency reversal agents with targeted cellular killing agents may be an effective approach to eradicating the viral reservoir., (© 2022. The Author(s).)

Determining how hematopoietic stem and progenitor cells (HSPCs) can be infected by viruses is necessary to understand and predict how the immune system will drive the host response. We present here a protocol to analyze the capacity of SARS-CoV-2 to infect different subsets of human HSPCs, inlcuding procedures for SARS-CoV-2 production and titration, isolation of human HSPCs from different sources (bone marrow, umbilical cord, or peripheral blood), and quantification of SARS-Cov-2 infection capacity by RT-qPCR and colony forming unit assay. For complete details on the use and execution of this protocol, please refer to Huerga Encabo et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Chronic viral infections are associated with hematopoietic suppression, bone marrow (BM) failure, and hematopoietic stem cell (HSC) exhaustion. However, how persistent viral challenge and inflammatory responses target BM tissues and perturb hematopoietic competence remains poorly understood. Here, we combine functional analyses with advanced 3D microscopy to demonstrate that chronic infection with lymphocytic choriomeningitis virus leads to (1) long-lasting decimation of the BM stromal network of mesenchymal CXCL12-abundant reticular cells, (2) proinflammatory transcriptional remodeling of remaining components of this key niche subset, and (3) durable functional defects and decreased competitive fitness in HSCs. Mechanistically, BM immunopathology is elicited by virus-specific, activated CD8 T cells, which accumulate in the BM via interferon-dependent mechanisms. Combined antibody-mediated inhibition of type I and II IFN pathways completely preempts degeneration of CARc and protects HSCs from chronic dysfunction. Hence, viral infections and ensuing immune reactions durably impact BM homeostasis by persistently decreasing the competitive fitness of HSCs and disrupting essential stromal-derived, hematopoietic-supporting cues., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Isringhausen et al.)

Aims: Haemophagocytosis in the bone marrow of patients who have succumbed to coronavirus disease 19 (COVID-19) has not been widely studied. The aims of the present study were to perform morphological analyses and morphometry of haemophagocytosis in the bone marrow of patients with severe COVID-19, and to correlate the findings with the clinical course of the disease., Methods and Results: In this single-centre study performed at the University Hospital Jena, bone marrow specimens of 15 deceased patients who had experienced a severe course of COVID-19 were sampled from the vertebral column during autopsy. Slides of the bone marrow were stained with routine stains or immunohistochemically, and further examined for haemophagocytosis by the use of light microscopy. To substantiate the morphological findings, additional slides were stained for CD163 and morphometry was performed. In all bone marrow samples, an increase in cellularity was found. Haemophagocytes with erythrophagocytosis were detected in 67% of the deceased patients. In tissues with low numbers of haemophagocytes or ill-defined haemophagocytes, an increase in iron deposits was frequently seen. Morphological findings were then correlated with several important clinical data, and the HScore (probability of having a reactive hemophagocytic syndrome) was calculated to posthumously confirm the diagnosis of secondary haemophagocytic lymphohistiocytosis. The median duration of disease and the hospitalisation time were lower in patients with haemophagocytosis (n = 10) than in patients without haemophagocytosis (n = 5). In addition, patients with haemophagocytes showed increased inflammatory parameters 2-5 days prior to death, in contrast to patients without haemophagocytes., Conclusions: Haemophagocytosis is a common finding in the bone marrow of deceased individuals with severe COVID-19, and may indicate fatal severe acute respiratory syndrome coronavirus 2 infections., (© 2020 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Background: Multiple myeloma (MM) remains an incurable disease and oncolytic viruses offer a well-tolerated addition to the therapeutic arsenal. Oncolytic reovirus has progressed to phase I clinical trials and its direct lytic potential has been extensively studied. However, to date, the role for reovirus-induced immunotherapy against MM, and the impact of the bone marrow (BM) niche, have not been reported., Methods: This study used human peripheral blood mononuclear cells from healthy donors and in vitro co-culture of MM cells and BM stromal cells to recapitulate the resistant BM niche. Additionally, the 5TGM1-Kalw/RijHSD immunocompetent in vivo model was used to examine reovirus efficacy and characterize reovirus-induced immune responses in the BM and spleen following intravenous administration. Collectively, these in vitro and in vivo models were used to characterize the development of innate and adaptive antimyeloma immunity following reovirus treatment., Results: Using the 5TGM1-Kalw/RijHSD immunocompetent in vivo model we have demonstrated that reovirus reduces both MM tumor burden and myeloma-induced bone disease. Furthermore, detailed immune characterization revealed that reovirus: (i) increased natural killer (NK) cell and CD8 + T cell numbers; (ii) activated NK cells and CD8 + T cells and (iii) upregulated effector-memory CD8 + T cells. Moreover, increased effector-memory CD8 + T cells correlated with decreased tumor burden. Next, we explored the potential for reovirus-induced immunotherapy using human co-culture models to mimic the myeloma-supportive BM niche. MM cells co-cultured with BM stromal cells displayed resistance to reovirus-induced oncolysis and bystander cytokine-killing but remained susceptible to killing by reovirus-activated NK cells and MM-specific cytotoxic T lymphocytes., Conclusion: These data highlight the importance of reovirus-induced immunotherapy for targeting MM cells within the BM niche and suggest that combination with agents which boost antitumor immune responses should be a priority., Competing Interests: Competing interests: MC is affiliated with Oncolytics Biotec Inc., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Background: Osteosarcoma is a malignant mesenchymal bone tumor. Although it is a common tumor in the appendicular skeleton of dogs and cats, it is rarely reported in birds. Retroviruses are usually associated with solid tumor development in different avian species., Case Presentation: This report aims to describe a case of osteosarcoma associated with the avian leukosis virus in a captive bare-faced curassow (Crax fasciolata). A captive adult female bare-faced curassow presented with lameness, hyporexia, and a non-ulcerative and firm tumor in the right femur. The bird was euthanized due to the poor prognosis. Histopathology revealed an infiltrative mesenchymal neoplasm consisting of spindle cells with moderate cell pleomorphism, organized in bundles and interspersed by marked deposition of the osteoid matrix, which was compatible with osteosarcoma affecting both femur and tibiotarsus, with renal metastasis. Immunohistochemistry of the primary and metastatic tumor demonstrated vimentin expression by neoplastic cells. Samples of the neoplasm, bone marrow, and spleen were processed for PCR, which enabled the demonstration of proviral avian leukosis virus (ALV) DNA., Conclusions: To the best of our knowledge, this is the first report of an osteosarcoma in a bare-faced curassow with an unusual polyostotic manifestation and associated with ALV infection.

Background: Hydroa Vacciniforme-like Lymphoproliferative Disorder (HV-LPD) is the name given to a group of Epstein-Barr virus (EBV)-associated diseases. It resembles hydroa vacciniforme (HV), the rarest form of photosensitivity, and is a T-cell disorder associated with an Epstein-Barr virus infection. The majority of diagnosed cases occur in East Asia and South America. It is rare in the United States and Europe. Multiple studies have revealed the clinical manifestation of an enlarged liver, but no gold standard such as pathology has yet supported this as a clinical sign of HV-LPD., Case Presentation: Here, we report a case of a 34-year-old Asian female with definite liver invasion. The patient had complained of a recurring facial rash for many years. The patient was admitted to the hospital because of an enlarged liver. After hospitalization, she was given an EB virus nucleic acid test. The EB virus nucleic acid test was positive, and pathological examination suggested that HV-LPD had invaded the skin, bone marrow, and liver. After being given antiviral treatment, the patient's symptoms were mitigated., Conclusions: Our case confirms the liver damage was caused by HV-LPD and the effectiveness of antiviral treatment.

Coronavirus disease 2019 (COVID-19) is a global public health emergency with many clinical facets, and new knowledge about its pathogenetic mechanisms is deemed necessary; among these, there are certainly coagulation disorders. In the history of medicine, autopsies and tissue sampling have played a fundamental role in order to understand the pathogenesis of emerging diseases, including infectious ones; compared to the past, histopathology can be now expanded by innovative techniques and modern technologies. For the first time in worldwide literature, we provide a detailed postmortem and biopsy report on the marked increase, up to 1 order of magnitude, of naked megakaryocyte nuclei in the bone marrow and lungs from serious COVID-19 patients. Most likely related to high interleukin-6 serum levels stimulating megakaryocytopoiesis, this phenomenon concurs to explain well the pulmonary abnormal immunothrombosis in these critically ill patients, all without molecular or electron microscopy signs of megakaryocyte infection.

A critical barrier to the development of a human immunodeficiency virus (HIV) cure is the lack of a scalable animal model that enables robust evaluation of eradication approaches prior to testing in humans. We established a humanized mouse model of latent HIV infection by transplanting "J-Lat" cells, Jurkat cells harboring a latent HIV provirus encoding an enhanced green fluorescent protein (GFP) reporter, into irradiated adult NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice. J-Lat cells exhibited successful engraftment in several tissues including spleen, bone barrow, peripheral blood, and lung, in line with the diverse natural tissue tropism of HIV. Administration of tumor necrosis factor (TNF)-α, an established HIV latency reversal agent, significantly induced GFP expression in engrafted cells across tissues, reflecting viral reactivation. These data suggest that our murine latency ("μ-Lat") model enables efficient determination of how effectively viral eradication agents, including latency reversal agents, penetrate, and function in diverse anatomical sites harboring HIV in vivo., (© 2020 Federation of American Societies for Experimental Biology.)

An effective strategy to cure HIV will likely require a potent and sustained antiviral T cell response. Here we explored the utility of chimeric antigen receptor (CAR) T cells, expressing the CD4 ectodomain to confer specificity for the HIV envelope, to mitigate HIV-induced pathogenesis in bone marrow, liver, thymus (BLT) humanized mice. CAR T cells expressing the 4-1BB/CD3-ζ endodomain were insufficient to prevent viral rebound and CD4 + T cell loss after the discontinuation of antiretroviral therapy. Through iterative improvements to the CAR T cell product, we developed Dual-CAR T cells that simultaneously expressed both 4-1BB/CD3-ζ and CD28/CD3-ζ endodomains. Dual-CAR T cells exhibited expansion kinetics that exceeded 4-1BB-, CD28- and third-generation costimulated CAR T cells, elicited effector functions equivalent to CD28-costimulated CAR T cells and prevented HIV-induced CD4 + T cell loss despite persistent viremia. Moreover, when Dual-CAR T cells were protected from HIV infection through expression of the C34-CXCR4 fusion inhibitor, these cells significantly reduced acute-phase viremia, as well as accelerated HIV suppression in the presence of antiretroviral therapy and reduced tissue viral burden. Collectively, these studies demonstrate the enhanced therapeutic potency of a novel Dual-CAR T cell product with the potential to effectively treat HIV infection.

Background: Nowadays, most blood products are leukocyte-reduced. After this procedure, the residual risk for transfusion transmitted cytomegalovirus (TT-CMV) is mostly attributed to cell-free viruses in the plasma of blood donors following primary infection or viral reactivation. Here, objectives are: 1) to study the behaviour of cell-free CMV through the blood component processing; 2) to determine the anti-CMV seroprevalence, the level of viremia, the window-period in blood donor population; and 3) to identify cases of TT-CMV in bone marrow transplant (BMT) recipients., Materials and Methods: Cell-free CMV was injected into blood bags originating from regular donors. Blood components were processed according to either the CompoSelect ® or the CompoFlow ® (Fresenius Kabi AG) techniques. Samples were analysed at each step for presence of virus DNA using quantitative polymerase chain reaction (PCR). The anti-CMV seroprevalence in our donor population was taken from our donor data system. The viremia was assessed in pooled plasmas samples from routine donations by quantitative PCR. Medical charts of 165 BMT anti-CMV seronegative recipients/anti-CMV seronegative donors who received CMV-unscreened blood products were reviewed., Results: Cell-free CMV passes without any decrease in viral load through all stages of blood processing. The anti-CMV seroprevalence was 46.13%. Four DNA positive samples out of 42,240 individual blood donations were identified (0.009%); all had low levels of viremia (range 11-255 IU/mL). No window-period donation was identified. No TT-CMV was found., Discussion: Cell-free CMV remains a concern with current blood component processing as it passes through all the processes. However, since low levels of CMV DNA were identified in the donations tested, and no BMT recipients had TT-CMV, the residual threat of TT-CMV after leukocyte reduction appears to be very low.

While feline leukemia virus (FeLV) has been shown to infect felid species other than the endemic domestic cat host, differences in FeLV susceptibility among species has not been evaluated. Previous reports have noted a negative correlation between endogenous FeLV (enFeLV) copy number and exogenous FeLV (exFeLV) infection outcomes in domestic cats. Since felids outside the genus Felis do not harbor enFeLV genomes, we hypothesized absence of enFeLV results in more severe disease consequences in felid species lacking these genomic elements. We infected primary fibroblasts isolated from domestic cats ( Felis catus ) and pumas ( Puma concolor ) with FeLV and quantitated proviral and viral antigen loads. Domestic cat enFeLV env and long terminal repeat (LTR) copy numbers were determined for each individual and compared to FeLV viral outcomes. FeLV proviral and antigen levels were also measured in 6 naturally infected domestic cats and 11 naturally infected Florida panthers ( P. concolor coryi ). We demonstrated that puma fibroblasts are more permissive to FeLV than domestic cat cells, and domestic cat FeLV restriction was highly related to enFeLV-LTR copy number. Terminal tissues from FeLV-infected Florida panthers and domestic cats had similar exFeLV proviral copy numbers, but Florida panther tissues have higher FeLV antigen loads. Our work indicates that enFeLV-LTR elements negatively correlate with exogenous FeLV replication. Further, Puma concolor samples lacking enFeLV are more permissive to FeLV infection than domestic cat samples, suggesting that endogenization can play a beneficial role in mitigating exogenous retroviral infections. Conversely, presence of endogenous retroelements may relate to new host susceptibility during viral spillover events. IMPORTANCE Feline leukemia virus (FeLV) can infect a variety of felid species. Only the primary domestic cat host and related small cat species harbor a related endogenous virus in their genomes. Previous studies noted a negative association between the endogenous virus copy number and exogenous virus infection in domestic cats. This report shows that puma cells, which lack endogenous FeLV, produce more virus more rapidly than domestic cat fibroblasts following cell culture challenge. We document a strong association between domestic cat cell susceptibility and FeLV long terminal repeat (LTR) copy number, similar to observations in natural FeLV infections. Viral replication does not, however, correlate with FeLV env copy number, suggesting that this effect is specific to FeLV-LTR elements. This discovery indicates a protective capacity of the endogenous virus against the exogenous form, either via direct interference or indirectly via gene regulation, and may suggest evolutionary outcomes of retroviral endogenization., (Copyright © 2020 American Society for Microbiology.)

Europe is currently experiencing a long-lasting African swine fever (ASF) epidemic, both in domestic pigs and wild boar. There is great concern that carcasses of infected wild boar may act as long-term virus reservoirs in the environment. We evaluated the tenacity of ASF virus (ASFV) in tissues and body fluids from experimentally infected domestic pigs and wild boar, which were stored on different matrices and at different temperatures. Samples were analysed at regular intervals for viral genome and infectious virus. ASFV was most stable in spleen or muscles stored at -20 °C and in blood stored at 4 °C. In bones stored at -20 °C, infectious virus was detected for up to three months, and at 4 °C for up to one month, while at room temperature (RT), no infectious virus could be recovered after one week. Skin stored at -20 °C, 4 °C and RT remained infectious for up to three, six and three months, respectively. In urine and faeces, no infectious virus was recovered after one week, irrespective of the matrix. In conclusion, tissues and organs from decomposing carcasses that persist in the environment for a long time can be a source of infection for several months, especially at low temperatures.

Pegiviruses frequently cause persistent infection (as defined by >6 months), but unlike most other Flaviviridae members, no apparent clinical disease. Human pegivirus (HPgV, previously GBV-C) is detectable in 1-4% of healthy individuals and another 5-13% are seropositive. Some evidence for infection of bone marrow and spleen exists. Equine pegivirus 1 (EPgV-1) is not linked to disease, whereas another pegivirus, Theiler's disease-associated virus (TDAV), was identified in an outbreak of acute serum hepatitis (Theiler's disease) in horses. Although no subsequent reports link TDAV to disease, any association with hepatitis has not been formally examined. Here, we characterized EPgV-1 and TDAV tropism, sequence diversity, persistence and association with liver disease in horses. Among more than 20 tissue types, we consistently detected high viral loads only in serum, bone marrow and spleen, and viral RNA replication was consistently identified in bone marrow. PBMCs and lymph nodes, but not liver, were sporadically positive. To exclude potential effects of co-infecting agents in experimental infections, we constructed full-length consensus cDNA clones; this was enabled by determination of the complete viral genomes, including a novel TDAV 3' terminus. Clone derived RNA transcripts were used for direct intrasplenic inoculation of healthy horses. This led to productive infection detectable from week 2-3 and persisting beyond the 28 weeks of study. We did not observe any clinical signs of illness or elevation of circulating liver enzymes. The polyprotein consensus sequences did not change, suggesting that both clones were fully functional. To our knowledge, this is the first successful extrahepatic viral RNA launch and the first robust reverse genetics system for a pegivirus. In conclusion, equine pegiviruses are bone marrow tropic, cause persistent infection in horses, and are not associated with hepatitis. Based on these findings, it may be appropriate to rename the group of TDAV and related viruses as EPgV-2., Competing Interests: The authors have declared that no competing interests exist.

Prior work demonstrated that epithelial V-like antigen (EVA), a cell surface adhesion molecule, is expressed in B lymphocytes and is necessary for the efficacy of anti-alpha 4 integrin treatment of experimental autoimmune encephalomyelitis (EAE), the mouse model of human multiple sclerosis. EVA deficiency is associated with a severe clinical phenotype of EAE in the presence or absence of treatment. Histological analysis revealed enhanced B cell-mediated autoimmunity and deposition of antibody and complement within the brain and spinal cord. Here our goal was to determine the molecular mechanism of EVA regulation of B lymphocyte function. Analysis of bone marrow from MOG-immunized mice revealed increased expansion of CD11c + B cells in EVA-deficient mice as compared to wild type controls. In vitro studies of mouse bone marrow B lymphocytes revealed enhanced proliferation of the CD11c + population in response to the Tlr7/8 agonist R848. An increase in R848-induced proliferation of CD11c + B cells was also seen in vitro in Daudi cells, a human B cell line, following knockdown of the mpzl2 gene that encodes EVA. These mechanisms were characterized further by global expression analysis of bone marrow from immunized EVA-deficient and wild type control mice. These data revealed increased expression of B cell associated genes and decreased expression of the anti-viral oligoadenylate synthase genes, Oas1 and Oas2, in the knockout condition. In Daudi cells, R848 treatment induced an increase in Oas2 expression in control cells that was not observed in EVA-deficient cells. EVA deficiency also was associated with increased transcription of an Epstein-Barr virus gene during lytic replication. These results suggest EVA expression and signaling prevent expansion of CD11c + B lymphocytes, a cellular phenotype associated with autoimmunity, increase expression of anti-viral oligoadenylate synthase genes, and reduce replication of a DNA virus., (Copyright © 2019. Published by Elsevier GmbH.)

We determined the temporal aspects of detecting bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in postmortem bone marrow samples of white-tailed deer ( Odocoileus virginianus ) using molecular and in vitro cell culture techniques. Bone marrow samples from carcasses were collected and assayed on the day of death and at intervals up to 16 wk after death. We recovered BTV and EHDV from fresh bone marrow collected at day 0 by isolation in Vero and BHK-21 cell cultures. However, attempts to replicate the viruses from aged bone marrow in Vero and BHK-21 cell cultures failed. The real-time quantitative reverse transcriptase PCR (qRT-PCR) results confirmed that EHDV and BTV can be detected in aged bone marrow for up to 12 and 16 wk, respectively, after death. The RNA of BTV and EHDV could be detected by qRT-PCR in white-tailed deer bone marrow for extended periods of time postmortem. This technique will provide a useful tool for retrospective determination of BTV or EHDV infection of white-tailed deer at the time of death.

Background: Disseminated Kaposi sarcoma (DKS) is present in patients with advanced HIV infection in whom co-infection with other opportunistic pathogens can occur. Bone marrow (BM) aspirate and biopsy comprise a robust diagnostic tool in patients with fever, cytopenias, and abnormal liver tests. However, the yield in patients with DKS has not been determined., Objective: The aim of this study was to evaluate the utility of BM aspirate and biopsy in patients with DKS., Methods: We included 40 male patients with a recent diagnosis of DKS. BM aspirate and biopsy was performed as part of the workup to rule out co-infections., Results: In four patients, Mycobacterium avium complex (MAC) was recovered from culture. In other four patients, intracellular yeasts were observed in the Grocott stain, diagnosed as Histoplasma. The yield of BM was calculated in 20%. Only 12 patients (30%) had fever and 11 (27.5%) had pancytopenia. Alkaline phosphatase (ALP) above normal values and C-reactive protein (CRP) were higher in patients with positive results for BM than in those with negative results (63% vs. 21.9%, and 3.0 vs. 1.2 mg/L; p = 0.03 in both comparisons). No differences were found when complete blood-count abnormalities were compared., Conclusion: We recommend performing a BM aspirate for stains, culture, and biopsy in all HIV patients with DKS, as this will permit the early diagnosis of co-infections and prevent further complications in those who receive chemotherapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cells sensing infection produce Type I interferons (IFN-I) to stimulate Interferon Stimulated Genes (ISGs) that confer resistance to viruses. During lympho-hematogenous spread of the mouse pathogen ectromelia virus (ECTV), the adaptor STING and the transcription factor IRF7 are required for IFN-I and ISG induction and resistance to ECTV. However, it is unknown which cells sense ECTV and which pathogen recognition receptor (PRR) upstream of STING is required for IFN-I and ISG induction. We found that cyclic-GMP-AMP (cGAMP) synthase (cGAS), a DNA-sensing PRR, is required in bone marrow-derived (BMD) but not in other cells for IFN-I and ISG induction and for resistance to lethal mousepox. Also, local administration of cGAMP, the product of cGAS that activates STING, rescues cGAS but not IRF7 or IFN-I receptor deficient mice from mousepox. Thus, sensing of infection by BMD cells via cGAS and IRF7 is critical for resistance to a lethal viral disease in a natural host., Competing Interests: The authors have declared that no competing interests exist.

Background: Feline leukemia virus (FeLV) is a serious viral infection in cats. FeLV is found in some tissues, such as spleen, lymph nodes and epithelial tissues. However, there is controversy about the organ in which the virus can be reliably detected in infected cats. The purpose of this study was to determine the level of viral infection in hemolymphatic tissues, including blood, bone marrow and spleen by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR)., Results: A total of 31 cats with clinical signs of FeLV infection associated with at least a single lineage hematologic cytopenia were included in this study. Peripheral blood, bone marrow and spleen samples were obtained from each cat. Complete blood counts, biochemical tests, and a rapid test to detect FeLV p27 antigen in blood samples of cats were performed. Of 31 cats, 9 had anemia alone, 4 had thrombocytopenia alone, 2 had neutropenia alone, 9 had bicytopenia of anemia and thrombocytopenia, 3 had bicytopenia of anemia and neutropenia, and 4 had pancytopenia. FeLV RNA was then detected by RT-qPCR in the whole blood, bone marrow and spleen. Viral RNA copy numbers were detected in all cats by RT-qPCR whereas 24 out of 31 cats were positive for the serum FeLV antigen. We detected a significantly greater number of viral RNA in the spleen compared with the whole blood and bone marrow., Conclusion: Spleen is a site where FeLV is most frequently detected in cats with hematologic cytopenias.

One of the most important clinical signs of dengue virus infection is the reduction of white blood cells and platelets in human peripheral blood (leukopenia and thrombocytopenia, respectively), which may significantly impair the clearance of dengue virus by the immune system. The cause of thrombocytopenia and leukopenia during dengue infection is still unknown, but may be related to severe suppression of bone marrow populations including hematopoietic stem cells and megakaryocytes, the progenitors of white blood cells and platelets respectively. Here, we explored the possibility that bone marrow suppression, including ablation of megakaryocyte populations, is caused by dengue virus infection of megakaryocytes. We used three different models to measure dengue virus infection and replication: in vitro, in a human megakaryocyte cell line with viral receptors, ex vivo, in primary human megakaryocytes, and in vivo, in humanized mice. All three systems support dengue virus infection and replication, including virus strains from serotypes 1, 2, and 3, and clinical signs, in vivo; all assays showed viral RNA and/or infectious viruses 7-14 days post-infection. Although we saw no significant decrease in cell viability in vitro, there was significant depletion of mature megakaryocytes in vivo. We conclude that megakaryocytes can produce dengue viruses in the bone marrow niche, and a reduction of cell numbers may affect bone marrow homeostasis., Competing Interests: The authors have declared that no competing interests exist.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infection that has recently emerged. This infectious disease is due to the transfer of SFTS virus (SFTSV) from the infected blood of animals to humans. Approximately 30% of patients who are infected with SFTS die from multiorgan failure associated with severe infection, systemic inflammatory response syndrome, or disseminated intravascular coagulation. We treated an elderly Japanese couple (husband and wife) who had genetically identical SFTSV infections and who both developed severe rhabdomyolysis., Case Presentation: An 80-year-old man presented to the clinic with a fever; his 74-year-old wife presented with a fever 9 days later. Their laboratory results at diagnosis showed severe rhabdomyolysis with significantly elevated creatinine kinase (detected levels: husband, 9546 U/L; wife, 15,933 U/L). The creatinine kinase isozyme was 100% MM type in both patients. In both the husband and wife, SFTSV was identified with real-time polymerase chain reaction analysis. The detected SFTSVs in both the husband and wife were identical according to the genome sequence analysis. The husband's bone marrow indicated macrophage activation syndrome, but he responded to supportive therapy. He was discharged after being hospitalized for 32 days. The wife was admitted to our hospital in critical condition 2 days after SFTS symptom onset. She died of multiorgan failure 8 days after onset, despite being cared for in an intensive care unit. Both of the patients presented with rhabdomyolysis following SFTS symptom onset. The patients' clinical outcomes were different from each other; i.e., the husband survived, and the wife died., Conclusions: SFTSV infection-associated rhabdomyolysis has been reported in one patient, and simultaneous onset in two related patients has not been described previously. Our findings suggest that similar biological responses occurred, but they resulted in different clinical outcomes in the patients infected by the identical SFTSV isolates. Notably, a patient's clinical outcome depends on their own immune response. We suggest that one component of viral rhabdomyolysis involves immune-mediated responses. Severe immunological responses may adversely affect the treatment outcome, as demonstrated by the wife's clinical course. Our findings demonstrate that a patient's immune response contributes to their prognosis following SFTSV infection.

Epstein-Barr virus (EBV) is associated with several disorders. EBV is known to modulate the proliferation and survival of hematopoietic cells such as B cells and T cells in human. However, the effects of EBV on hematopoiesis itself have not been investigated. To study EBV infection in murine models, their hematopoiesis must be humanized, since EBV infection is limited only in primates. To engraft the human hematopoiesis, NOD/Shi-scid-IL2rγ null (NOG) mice were used. Usually, the hematopoiesis humanized mice reconstitute only lymphoid cells, but myeloid cells are not. However, we revealed human macrophages (hMφ) and their precursor monocytes were increased in peripheral tissues of EBV-infected mice. Furthermore, our previous report indicated Mφ accumulation in spleen was essential for development of EBV-positive tumors, suggesting that EBV modulates human hematopoiesis in order to thrive. Interestingly, we revealed a dramatic increase of immature granulocytes only in bone marrow of EBV-infected mice. In addition, GM-CSF, a cytokine that is essential for differentiation of the myeloid lineage, was significantly increased in EBV-infected mice. These results were also reproduced in patients with EBV-related disorders. We suggest that the hematopoietic alterations during EBV-infection might contribute immune suppression to the development and exacerbation of EBV-related disorders.

Viral rebound upon stopping combined antiretroviral therapy poses a major barrier toward an HIV cure. Cellular and anatomical sources responsible for reinitiating viral replication remain a subject of ardent debate, despite extensive research efforts. To unravel the source of rebounding viruses, we conducted a large-scale HIV-STAR (HIV-1 sequencing before analytical treatment interruption to identify the anatomically relevant HIV reservoir) clinical trial. We collected samples from 11 participants and compared the genetic composition of (pro)viruses collected under treatment from different cellular and anatomical compartments with that of plasma viruses sampled during analytical treatment interruption. We found a remarkably heterogeneous source of viral rebound. In addition, irrespective of the compartment or cell subset, genetically identical viral expansions played a significant role in viral rebound. Our study suggests that although there does not seem to be a primary source for rebound HIV, cellular proliferation is an important driver of HIV persistence and should therefore be considered in future curative strategies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Infectious Bursal Disease Virus (IBDV) of the ITA genotype (G6) was shown to have peculiar molecular characteristics and, despite a subclinical course, aggressiveness towards lymphoid tissues after experimental infection of specific-pathogen-free (SPF) chickens. The aim of the present study was to evaluate and compare with a Classical IBDV strain, ITA IBDV distribution and persistence in various tissues (bursa of Fabricious, spleen, thymus, bone marrow, caecal tonsils, Harderian gland, kidney, liver and proventriculus), its cloacal shedding and the involvement of gut TLR-3 in duodenum tissues. The 35-day-old SPF chickens were experimentally infected and sampled up to 28 days post infection (dpi) for IBDV detection and TLR-3 quantification by qRT-PCR. The ITA IBDV strain was detected in lymphoid and most non-lymphoid tissues up to the end of the trial, with higher loads compared to the Classical IBDV. Most of those differences were found during the first 2 weeks post-infection. Notably, bone marrow and caecal tonsils presented higher viral loads until 28 dpi, allowing to speculate that these organs may serve as non-bursal lymphoid tissues supporting virus replication. Differences in relative TLR-3 gene expression between ITA IBDV-infected birds and Classical-IBDV infected ones were observed at 4, 14 and 21 dpi, being initially higher in Classical group and later in ITA group. Our results provide new insights into IBDV pathogenesis showing that IBDV of ITA genotype leads to a high and persistent viral load in lymphoid tissues and to a delayed antiviral response., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Myxovirus resistance 1 ( Mx1 ) is an interferon-induced gene that encodes a GTPase that plays an important role in the defense of mammalian cells against influenza A and other viruses. The Mx1 protein can restrict a number of viruses independently of the expression of other interferon-induced genes. Mx genes are therefore considered to be an important part of the innate antiviral immune response. However, the possible impact of Mx expression in the hematopoietic cellular compartment has not been investigated in detail in the course of a viral infection. To address this, we performed bone marrow chimera experiments using congenic B6.A2G Mx1 +/+ and B6.A2G Mx1 -/- mice to study the effect of Mx1 expression in cells of hematopoietic versus nonhematopoietic origin. Mx1 +/+ mice were protected and Mx1 -/- mice were susceptible to influenza A virus challenge infection, regardless of the type of bone marrow cells ( Mx1 +/+ or Mx1 -/- ) the animals had received. Infection with Thogoto virus, however, revealed that Mx1 -/- mice with a functional Mx1 gene in the bone marrow compartment showed reduced liver pathology compared with Mx1 -/- mice that had been grafted with Mx1 -/- bone marrow. The reduced pathology in these mice was associated with a reduction in Thogoto virus titers in the spleen, lung, and serum. Moreover, Mx1 +/+ mice with Mx1 -/- bone marrow failed to control Thogoto virus replication in the spleen. Mx1 in the hematopoietic cellular compartment thus contributes to protection against Thogoto virus infection. IMPORTANCE Mx proteins are evolutionarily conserved in vertebrates and can restrict a wide range of viruses in a cell-autonomous way. The contribution to antiviral defense of Mx1 expression in hematopoietic cells remains largely unknown. We show that protection against influenza virus infection requires Mx1 expression in the nonhematopoietic cellular compartment. In contrast, Mx1 in bone marrow-derived cells is sufficient to control disease and virus replication following infection with a Thogoto virus. This indicates that, in addition to its well-established antiviral activity in nonhematopoietic cells, Mx1 in hematopoietic cells can also play an important antiviral function. In addition, cells of hematopoietic origin that lack a functional Mx1 gene contribute to Thogoto virus dissemination and associated disease., (Copyright © 2019 American Society for Microbiology.)

Epstein-Barr virus (EBV) infection is associated with pathogenesis of various cancers, including extranodal natural killer/T-cell lymphoma, nasal type (ENKL). ENKL tumor cells are positive for EBV-encoded RNA1 (EBER1), which is the most useful marker to identify ENKL tumor cells in histopathology. Currently, EBER1 in situ hybridization (ISH) is recommended to evaluate bone marrow (BM) involvement of ENKL. However, the actual burden of EBER1-positive cells in normal BM specimens remains unclear. In the present study, we performed EBER1 ISH on 111 BM specimens, which were obtained during an initial staging procedure in patients with EBV-negative cancers and were also negative for BM involvement. One or more EBER1-positive cells per whole specimen were observed in 38 specimens (34%). The number of EBER1-positive cells was distributed as follows: single positive cell, n = 17; two positive cells, n = 13; three positive cells, n = 3; and four positive cells, n = 5. These findings suggest that four or fewer EBER1-positive cells can be observed in BM specimens of patients with non-EBV-related cancers. The clinical implications of a small number of EBER1-positive cells in BM specimens of patients with ENKL should be evaluated in further studies., (© 2019 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Objective: To determine the incidence of positive CMV antigenemia (CMV-Ag) in patients with autoimmune rheumatic diseases (AIRD) and to describe the outcomes of these patients., Methods: From January 2011 to December 2014, a total of 443 patients with AIRD were enrolled in this retrospective analysis. Demographic, clinical and laboratory data, current clinical manifestations, organs affected by CMV infection, therapeutic management and outcomes were evaluated. The CMV-Ag was considered positive when one cell was detected at least., Results: CMV-Ag was requested in 70 (15.8%) patients with suspicious CMV infection and was positive in 24 (34.3%). The incidence rate of positive CMV-Ag was 4.97% (95% CI 3.1-7.4%). Systemic lupus erythematosus (SLE) (59%), followed by ANCA-related vasculitis (18.2%) and rheumatoid arthritis (9%) were the diseases more associated with positive CMV-Ag. At the time of CMV infection, SLE patients had moderate to severe disease activity, with high frequency of positive anti-dsDNA antibody (69.2%) and complement consumption (61.5%), as well as high doses of corticosteroids and use of immunosuppressants. The main CMV sites involved were lung (45.5%), bone marrow (40.9%) and gut (27.3%). Mortality rate was 45.5%, especially in those with higher doses of daily oral corticosteroids (107 ± 55.4 mg vs. 71.7 ± 46.3 mg; p = 0.07) and lower number of lymphocytes (309 ± 368.2/mm 3 vs. 821 ± 692.9/mm 3 ; p = 0.06)., Conclusions: Our data showed high incidence of CMV-Ag in AIRD patients, particularly those with SLE and greater disease severity. In addition, it was observed high mortality in these patients, highlighting the CMV infection should be included in differential diagnosis.

Red bone marrow is physiologically unique in that it is both the major hematopoietic organ and a primary lymphoid organ. Porcine reproductive and respiratory syndrome virus (PRRSV) affects normal bone marrow functions. The cumulative effect of PRRSV infection is acute bone marrow failure, i.e., hypoplasia characterized by the absence of normal myeloid and erythroid precursors and increased red bone marrow M:E ratios. The measurable clinical consequence of PRRSV infection on normal red bone marrow functions is a reduction in the number of cells emigrating to the peripheral blood resulting in leucopenia, anemia, and thrombocytopenia. These observations may be explained by the fact that bone marrow-derived mononuclear cells, i.e., imDCs, mDCs, monocytes, macrophages, and myeloid precursor cells are susceptible to PRRSV. Apoptosis in bone marrow-derived cells occurs both as a direct consequence of infection and indirectly via a bystander effect. Immunologically, PRRSV-susceptible mononuclear cells are the first line of defense against microbial infection and responsible for antigen recognition, processing, and presentation to T and B cells; a critical step in the initiation and development of an effective adaptive immune. Thus, impairment of normal immune function renders the host less able to resist and/or eliminate secondary infectious agents and partially explains the synergy between PRRSV and bacterial and viral co-infections., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Parvovirus B19 (PVB19) commonly infects children and is usually asymptomatic. Lethal outcomes of PVB19 infection are unusual; nevertheless, the two cases reported here are rare examples of PVB19-induced hemophagocytic syndrome and myocarditis in infants and children. The two cases show the indisputable usefulness of immunohistochemistry and in situ hybridization in the detection of PVB19. In the death investigations, histopathological examinations provided stronger evidence than did serology or molecular biology. The cases also highlight the importance of forensic autopsy in vaccine-related death. As vaccine-related deaths are what people fear and may cause declines in vaccination rates, it is important to clarify deaths temporally or causally associated with vaccine administration.

Human immunodeficiency virus (HIV) is associated with an increased risk of developing Hodgkin lymphoma (HL). South Africa (SA) has the highest HIV prevalence rate in the world. There is currently no outcome-based data for HIV-associated HL from SA. A bone marrow database was compiled of all bone marrow biopsies (BMB) reported at National Health Laboratory Service (NHLS) Groote Schuur Hospital (GSH) between January 2005 and December 2012. Patients who had a BMB performed for staging of HL or where HL was diagnosed on the BMB were included for further analysis. Clinical and laboratory data was extracted from medical and laboratory records. Primary outcome measures included histological subtype, bone marrow infiltration (BMI) by HL, CD4 count, HIV-viral load (HIV-VL), tuberculosis (TB) data, treatment with chemotherapy and 5-year overall survival (OS). The database included 6569 BMB and 219 patients of these had HL and were included for analysis. The median age at presentation (32 years) was similar in the HIV+ and HIV- populations. While males predominated in the HIV- group, females predominated in the HIV+ group (male:female ratio of 1.5:1 vs 0.7:1, respectively). The majority of patients (71%) were HIV negative (HIV-) and 29% were HIV positive (HIV+). The diagnosis of HL was made on BMB in 17% of cases. BMI was seen in 37% (82/219) overall, and was found in more HIV+ patients (61%; 39/64) than HIV- patients (28%; 43/155; p = 0.03). The histological subtype varied according to HIV status with nodular sclerosis classical Hodgkin lymphoma (NSCHL) being most frequent in the HIV- group and classical Hodgkin lymphoma (CHL)-unclassifiable the most frequent in the HIV+ group. HIV+ patients had a median CD4 count of 149 × 10 6 /L and 39% were anti-retroviral therapy (cART) naive at HL diagnosis. HIV+ patients had received anti-TB therapy more frequently than HIV- patients (72% vs 17%; p = 0.007). More HIV+ patients did not receive chemotherapy than HIV- patients (31% vs 3%; p = 0.001). The 5-year OS was 56%. HIV+ patients with BMI had a 5-year OS of 18%. BMI, HIV status, low CD4 count, histological subtype and TB therapy had a statistical significant impact on 5-year OS (p < 0.01). The 5-year OS was 56%, with both BMI and HIV+ status being associated with poor survival. BMB provided the diagnosis of HL in 17% of cases, confirming its diagnostic utility in our setting. Our cohort showed similar survival outcomes to other countries in Africa, Asia and Central America with comparable socio-economic constraints to SA.

Lentiviral vectors are the method of choice for stable gene modification of a variety of cell types. However, the efficiency with which they transduce target cells varies significantly, in particular their typically poor capacity to transduce primary stem cells. Here we describe the isolation and enrichment of murine bone-marrow mesenchymal stem cells (MSCs) via fluorescence-activated cell sorting (FACS); the cloning, production, and concentration of high-titer second generation lentiviral vectors via combined tangential flow filtration (TFF) and ultracentrifugation; and the subsequent high-efficiency gene modification of MSCs into insulin-producing cells via overexpression of the furin-cleavable human insulin (INS-FUR) gene.

Various infectious diseases can hyper-stimulate the immune system, causing hemophagocytic syndrome (HPS). Little is known regarding the accuracy of diagnostic criteria and epidemiological triggering factors in the acquired immunodeficiency syndrome (AIDS) setting. We investigated the major infectious disease triggers of HPS in patients living with human immunodeficiency virus (HIV)/AIDS and determined the accuracy of bone marrow aspiration (BMA). The inclusion criteria were (i) confirmed HIV diagnosis, (ii) bone marrow aspiration, and (iii) a minimum of four HPS criteria. Patients were further classified into those with four presumed HPS criteria, or ≥ 5 confirmed criteria. The disease triggers, accuracy of bone marrow aspiration, and prognosis markers were examined. Presumed HPS was observed in 15/36 patients (41%), and confirmed HPS in 58% (n = 21). The major etiological triggers were infection with Mycobacterium (34%), Cytomegalovirus (14%), Cryptococcus neoformans (11%), and hematological or tumoral disease (11%). BMA demonstrated 93% specificity on screening diagnosis (odds ratio [OR] 12.7, 95% confidence interval [CI] 1.4-115.1, P = 0.01). Ferritin > 5000 ng/mL correlated with probability of death in univariate analysis (OR 6.00, 95% CI 1.33-27.05, P = 0.02). Ferritin performance as test of death probability presented area under the curve as 0.74 (95% CI 0.56-0.91, P = 0.016). However, neither cluster of differentiation for lymphocyte count nor HIV viral load correlated with patient deaths. Mycobacterium spp. and Cytomegalovirus were the main factors triggering HPS, followed by Cryptococcus neoformans, and hematological and tumoral diseases. High ferritin levels were associated with increased death probability. High specificity was noted with BMA.

Ebola virus (EBOV) and Marburg virus (MARV) outbreaks are highly lethal, and infection results in a hemorrhagic fever with complex etiology. These zoonotic viruses dysregulate the immune system to cause disease, in part by replicating within myeloid cells that would normally innately control viral infection and shape the adaptive immune response. We used triple knockout (TKO)-bone marrow, liver, thymus (BLT) humanized mice to recapitulate the early in vivo human immune response to filovirus infection. Disease severity in TKO-BLT mice was dissimilar between EBOV and MARV with greater severity observed during EBOV infection. Disease severity was related to increased Kupffer cell infection in the liver, higher levels of myeloid dysfunction, and skewing of macrophage subtypes in EBOV compared with MARV-infected mice. Overall, the TKO-BLT model provided a practical in vivo platform to study the human immune response to filovirus infection and generated a better understanding of how these viruses modulate specific components of the immune system.

Japanese Encephalitis Virus (JEV) is a flavivirus responsible for an important zoonotic, vector-borne disease included in the OIE list. JEV is endemic in a large area of Asia. In Italy, JEV has been found in dead birds collected in 1997-2000 and in a pool of Culex pipiens mosquitoes collected in 2010. Viral ecology in the inter-epidemic periods is not known. The aim of this study was to investigate JEV in FFPE archival samples of healthy birds collected in 1997-2000 in Tuscany (Italy) in the same area and a few months after collection of birds resulted infected by JEV. Different samples from 37 young birds and 83 adults were available. Immunohistochemistry detected JEV antigen only in bone marrow samples from 12 young healthy birds. Positive cells were morphologically referable to monocyte-macrophages lineage and were positive for anti-CD11b in serial sections. Real-time PCR detected JEV RNA in four of these samples. These results suggest that healthy birds can harbour JEV in bone marrow cells, while no other organs resulted infected. The role of healthy birds in JEV ecology should be better investigated. Surveillance programmes should include sampling of the most appropriate target organs., (© 2018 Blackwell Verlag GmbH.)

Hepatitis E virus (HEV) transmission through infected blood and blood products has already been described. However, little is known about the bone marrow (BM) as source of HEV infection. Our study aimed to investigate the presence of HEV antigen (Ag) and histological changes in BM of cynomolgus monkeys (Macaca fascicularis) experimentally and naturally infected with HEV. Four cynomolgus monkeys with acute, and two with chronic hepatitis E ─ after immunosuppressive therapy with tacrolimus ─ were compared with one colony-bred animal naturally infected. Both, natural and experimental infections were characterized by anti-HEV IgG seroconversion detected by ELISA, and viral RNA isolation confirmed by RT-qPCR and qualitative nested RT-PCR. BM biopsies were collected from all animals, submitted to histology and indirect immunofluorescence techniques and observed, respectively, by light and confocal microscopy. The HEV Ag-fluorescent-labeled cells were detected from BM biopsies obtained from three monkeys with acute and one with chronic hepatitis E, and also from the naturally infected monkey. In the experimentally infected animals with acute hepatitis, HEV Ag detection occurred at 160 days post-infection, even after viral clearance in serum, feces, and liver. Double-stranded RNA, a replicative marker, was detected in BM cells from both acute and chronically infected animals. Major histological findings included vacuolization in mononuclear and endosteal cells, an absence of organized inflammatory infiltrates, and also some fields suggesting displasic focal BM disease. These findings support the hypothesis of BM cells as secondary target sites of HEV persistence. Further experimental studies should be carried out to confirm the assumption of HEV transmission through BM transplantation., Competing Interests: The authors have declared that no competing interests exist.

Viral RNA (vRNA) is found in mice inoculated with coxsackievirus-B4E2 (CV-B4E2). The CV-B4E2 infection of murine spleen cells in vitro is enhanced with CV-B4E2-infected mouse serum. It has been investigated whether monocyte/macrophages were targets of CV-B4E2 in mice. vRNA has been detected in spleen and bone marrow of infected animals. The levels of vRNA were higher in CD14+ cells than in CD14- spleen cells and in F4/80- cells than in F4/80+ spleen cells. Meanwhile, CD14+ cells and F4/80- cells were more permissive to CV-B4E2 in vitro and the infection was enhanced when the virus was mixed with immune serum. While CV-B4E2 infected BMDM cultures (98% F4/80+); however, the immune serum did not enhance the infection. In conclusion, CV-B4E2 infects monocytes (CD14+, F4/80-) and macrophages (CD14+, F4/80+) in vivo and immune serum can enhance the in vitro infection of these cells arising out of the spleen., (Copyright © 2018 Elsevier Inc. All rights reserved.)